Method and kit for detection of microorganism

ABSTRACT

Live cells of microorganism in a test sample are detected by distinguishing the live cells from dead cells or injured cells by the following steps of:
         a) adding an agent capable of covalently binding to DNA or RNA by irradiation with light having a wavelength of 350 nm to 700 nm to the test sample;   b) irradiating the test sample to which the cross-linking agent is added with light having a wavelength of 350 nm to 700 nm;   c) amplifying a target region of DNA or RNA of the microorganism contained in the test sample by a nucleic acid amplification method in the presence of an agent for suppressing an action of a nucleic acid amplification inhibitory substance, without extracting nucleic acids from the cells; and   d) analyzing the amplified product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase under 35 U.S.C. §371 ofInternational Application PCT/JP2010/062474, filed Jul. 23, 2010, whichwas published in a non-English language, which claims priority to JPApplication No.: 2009-173566, filed Jul. 24, 2009.

TECHNICAL FIELD

The present invention relates to a method and kit for detecting amicroorganism contained in a foodstuff, a biological sample and anenvironmental sample such as industrial water and tap water. Moreprecisely, the present invention relates to a method and kit fordetection of a microorganism that enable selective detection of livecells of a microorganism contained in a foodstuff, a biological sample,a swab sample, and an environmental sample such as industrial water andtap water.

BACKGROUND ART

The plate culture method has been conventionally used for measurement oftotal live bacterial counts in foodstuffs, biological samples, swabsamples, or environmental samples. However, the plate culture methodrequires time of about two days to one month to obtain a result.

Because of the improvements of sterilization techniques and processingtechniques for foodstuffs, needs for distinguishing live states ofmicroorganisms from dead states of microorganisms existing in testsamples are increasing even for the cases where the cells exist in anextremely small amount. In the fields of food sanitation inspection andclinical test, in particular, as a quick method for detecting bacteria,it is attempted to determine presence or absence of bacteria or quantifybacteria by amplifying genes specific to the bacteria by PCR to such anamount that the genes can be visually observed. However, if a bacterialDNA is targeted, the background of dead cells originally contained inthe test sample is also detected, and therefore a positive resultobtained by PCR does not necessarily suggest the presence of livebacteria. Therefore, the current situation in the fields of foodsanitation and clinical test is that PCR is not used widely, although itis a highly sensitive and quick technique.

In these days, it is attempted to detect and quantify only live cells ofmicroorganism in a test sample by preparing cDNA with reversetranscriptase for mRNA as a target and performing PCR with primersspecific to various bacteria. However, in this method, the reversetranscription of mRNA of dead cells itself is not inhibited, and when10⁴ cfu/ml or 10⁴ cfu/g or more of dead cells are contained in the testsample, background of the dead cells is detected. Therefore, this methodcannot be said to be sufficient as a method for determining the live anddead states.

Specifically, as a method for distinguishing live state from dead statesof microorganisms such as bacteria using the PCR method, the methodsdescribed in Patent document 1 and 2 have been disclosed. However, thefollowing problems remain in these methods for distinguishing live anddead states of microorganisms such as bacteria using the PCR method.

As for the technique disclosed in Patent document 1, examples arementioned for distinction of dead cells contained in boiled foodstuffssubjected to high temperature long time sterilization at 100° C. for 10to 30 minutes, and microorganisms contained in foodstuffs subjected toethanol sterilization or formaldehyde sterilization. However, especiallythe treatment of the latter type, there are not foodstuffs actuallysubjected to such pasteurization treatments. Moreover, there are notsupposed detection of only live microorganisms in foodstuffs subjectedto the currently major sterilization method in the food industry, lowtemperature long time pasteurization (LTLT pasteurization), hightemperature short time pasteurization (HTST pasteurization), or ultrahigh temperature pasteurization (UHT pasteurization), and detection ofonly live specific pathogenic bacteria in clinical specimens ofinfectious disease patients administered with antibiotics. Moreover, inthe case of a test sample of a foodstuff or clinical specimen containingdead cells background at a concentration of 10⁴ cfu/ml or higher, theamounts of the final PCR amplified products derived from dead cellsexceed the detection limit of the technique of Patent document 1, andtherefore it is impossible to determine whether a positive response of atest sample obtained by PCR is derived from live cells or dead cells.

Further, as the technique of Patent document 2, disclosed is a method ofdistinguishing live cells from dead cells by utilizing relative decreasein RNA/DNA molar ratio of dead cells compared with that of live cells.In this method, the total RNA is extracted, complementary DNA isprepared by using a reverse transcription reaction, then PCR isperformed to calculate the Ct value thereof, and the molar concentrationof RNA is obtained by using a separately prepared calibration curve.Separately, a region of chromosomal DNA corresponding to that RNA isamplified by PCR to obtain the Ct value thereof, and the molarconcentration of the chromosomal DNA is calculated on the basis of thecalibration curve to obtain the RNA/DNA molar ratio. That is, the aboveprocedure requires to perform troublesome extraction of total RNA anduses two steps of reverse transcription reaction and PCR. Therefore,this technique is inferior to usual PCR targeting DNA in quantificationperformance and quickness. Further, RNA is continuously produced in livecells, whereas RNA derived from dead cells is decomposed over time at anearly stage. Therefore, the technique lacks stability. Furthermore, in afoodstuff or clinical specimen containing dead cells at a highconcentration, only live cells of 1/10 of that concentration can bedetected by this technique. Therefore, it is difficult to apply thistechnique in the fields of food sanitation inspection and clinical test,which require quickness, high sensitivity and accuracy.

A method for selectively detecting live cells (Viable-and-Culturablecells) of a microorganism by distinguishing them from dead cells orinjured cells (Viable-but-Non Culturable cells (VNC cells)) is disclosedin Patent document 3. The method disclosed in Patent document 3 is amethod comprising the step of treating a test sample with atopoisomerase poison and/or a DNA gyrase poison, the step of extractingDNA from the test sample, and amplifying a target region of theextracted DNA by PCR, and the step of analyzing an amplified product,and as the topoisomerase poison or the DNA gyrase poison, ethidiummonoazide is exemplified.

A method in which ethidium monoazide is used is also disclosed inNon-patent document 1. This method is a detection method comprising thestep of adding ethidium monoazide to a test sample and irradiating thesample with light, the step of extracting DNA from the sample after theirradiation, and the step of amplifying a specific region by PCR usingthe extracted DNA as a template. Moreover, a technique ofsemi-quantitatively quantifying live cells count by a combination ofculture of a microorganism and real-time PCR is disclosed in Non-patentdocument 1.

Moreover, as a method for still more clearly distinguishing live cellsand injured cells of a microorganism, the method described in Patentdocument 4 is disclosed. This method is a method comprising the step ofadding a cross-linker capable of cross-linking a DNA by irradiation withlight having a wavelength of 350 nm to 700 nm to a test sample, the stepof irradiating the test sample to which the cross-linker is added withlight having a wavelength of 350 nm to 700 nm, the step of removing thecross-linker contained in the test sample irradiated with light, thestep of adding a medium to the test sample from which the cross-linkeris removed and incubating the test sample, the step of adding again thecross-linker capable of cross-linking a DNA by irradiation with lighthaving a wavelength of 350 nm to 700 nm to the incubated test sample,the step of irradiating the test sample to which the cross-linker isadded with light having a wavelength of 350 nm to 700 nm, the step ofextracting a DNA from the test sample and amplifying a target region ofthe extracted DNA by a nucleic acid amplification method, and the stepof analyzing the amplified product.

Meanwhile, there is suggested a possibility that, in amplification ofnucleic acid by PCR, albumin may suppress inhibition activity of a PCRinhibitor, or promote the reactions of PCR (Non-patent document 2).Moreover, it is also suggested that calcium inhibits the reactions ofPCR, but the inhibition of PCR by calcium can be made tolerable byaddition of magnesium ions (Non-patent document 3).

Moreover, there is disclosed a method of performing reactions of PCRusing a bacterial DNA as a template, in which the reactions of PCR areperformed without extracting the DNA from the bacterium (Non-patentdocument 4, Patent document 5). In Patent document 5, there is disclosedthat random PCR is performed in a bacterium in the DNA fingerprintingmethod, and phosphates and dodecylsulfates are mentioned as componentsof the buffer composition for nucleic acid synthesis.

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: Domestic Laid-Open Publication of a Japanese    translation of PCT Application (KOHYO) No. 2003-530118-   Patent document 2: International Patent Publication WO2002/052034-   Patent document 3: International Patent Publication WO2007/094077-   Patent document 4: International Patent Publication WO2009/022558-   Patent document 5: International Patent Publication WO2004/104196

Non-patent documents

-   Non-patent document 1: Rudi, K., et al., Letters in Applied    Microbiology, 2005, Vol. 40, pp. 301-306-   Non-patent document 2: Forbes, B. E., et al., Journal of Clinical    Microbiology, 1996, 34 (9), pp. 2125-2128-   Non-patent document 3: Bickley, J., et al., Letter in Applied    Microbiology, 1996, 22, pp. 153-158-   Non-patent document 4: Kimberly, A., et al., BioTechniques, 31,    2001, pp. 598-607

DISCLOSURE OF THE INVENTION Problems to be Resolved by the Invention

By the aforementioned method using a topoisomerase poison and/or a DNAgyrase poison, or a cross-linker, live cells of microorganism,especially live cells of Klebsiella, Citrobacter, Listeria, Salmonellabacteria and so forth can be selectively detected with high sensitivity.However, a further improved method, especially a method for highlysensitively or highly accurately detecting live cells of Escherichia orSalmonella bacteria has been desired.

An object of the present invention is to provide a novel method for moreselectively detecting live cells of a microorganism contained in afoodstuff or biological sample compared with dead cells or injured cellsof the microorganism, and a kit for performing such a method.

Means of Solving the Problems

The inventors of the present invention have made extensive studies on amethod of discriminating between life and death of microorganisms, whichis applicable to various sterilization methods and is suitable for foodsanitation inspections of high detection sensitivity, and on a method ofdetecting a specific pathogen in a patient with an infection in hospitalor clinical practice. As a result, the inventors have found out that thedistinction can be attained with high sensitivity by adding an agentcapable of covalently binding to DNA or RNA by irradiation with lighthaving a wavelength of 350 nm to 700 nm to a test sample; irradiatingthe test sample with light having a wavelength of 350 nm to 700 nm;adding an agent for suppressing an action of a nucleic acidamplification inhibitory substance, a magnesium salt, and an organicacid salt or a phosphoric acid salt, and amplifying a chromosomal DNA ofa microorganism flowing out of cells by a nucleic acid amplificationreaction. Thus, the present invention has been completed.

That is, the present invention provides a method for detecting livecells of a microorganism in a test sample by distinguishing the livecells from dead cells or injured cells, which comprises the steps of:

a) adding an agent capable of covalently binding to a DNA or RNA byirradiation with light having a wavelength of 350 nm to 700 nm to thetest sample;

b) irradiating the test sample to which the agent is added with lighthaving a wavelength of 350 nm to 700 nm;

c) amplifying a target region of the DNA or RNA of the microorganismcontained in the test sample by a nucleic acid amplification method inthe presence of an agent for suppressing an action of a nucleic acidamplification inhibitory substance, without extracting nucleic acidsfrom the cells; and

d) analyzing the amplified product.

In a preferred embodiment of the present invention, the amplification ofthe target region is performed in microbial cells.

In a preferred embodiment of the aforementioned method, in theaforementioned step c), the amplification of the target region isperformed in the presence of one or more kinds selected from asurfactant, a magnesium salt, and an organic acid salt or a phosphoricacid salt.

In a preferred embodiment of the aforementioned method, before theaforementioned step c), the steps a) and b) are repeatedly performed.

In a preferred embodiment of the aforementioned method, before theaforementioned step a), the following step e) is performed:

e) treating the test sample with an enzyme having an activity ofdecomposing cells other than that of microorganism, a colloidal particleof a protein, a lipid, or a saccharide existing in the test sample.

In a preferred embodiment of the aforementioned method, the enzyme isselected from a protease, a lipid-degrading enzyme and asaccharide-degrading enzyme.

In a preferred embodiment of the aforementioned method, the test sampleis any one of a foodstuff, a biological sample, drinking water,industrial water, environmental water, wastewater, soil and a swabsample.

In a preferred embodiment of the aforementioned method, themicroorganism is a bacterium or a virus.

In a preferred embodiment of the aforementioned method, the bacterium isa gram-negative bacterium.

In a preferred embodiment of the aforementioned method, the agentcapable of covalently binding to a DNA or RNA by irradiation with lighthaving a wavelength of 350 nm to 700 nm is selected from ethidiummonoazide, ethidium diazide, propidium monoazide, psoralenpsoralen,4,5′,8-trimethylpsoralen, and 8-methoxypsoralenpsoralen.

In a preferred embodiment of the aforementioned method, the agent forsuppressing an action of a nucleic acid amplification inhibitorysubstance consists of one or more kinds selected from albumin, dextran,T4 gene 32 protein, acetamide, betaine, dimethyl sulfoxide, formamide,glycerol, polyethylene glycol, soybean trypsin inhibitor,α2-macroglobulin, tetramethylammonium chloride, lysozyme, phosphorylase,and lactate dehydrogenase.

In a preferred embodiment of the aforementioned method, the organic acidsalt is selected from an acetic acid salt, a propionic acid salt and acitric acid salt.

In a preferred embodiment of the aforementioned method, the phosphoricacid salt is a pyrophosphoric acid salt.

In a preferred embodiment of the aforementioned method, the targetregion is a target region of 50 to 5,000 nucleotides.

In a preferred embodiment of the aforementioned method, the targetregion is a target region corresponding to a gene selected from 5S rRNAgene, 16S rRNA gene, 23S rRNA gene, and tRNA gene of the DNA of the testsample.

In a preferred embodiment of the aforementioned method, the nucleic acidamplification method is PCR, LAMP, SDA, LCR, TMA, TRC, HC, or themicroarray method.

In a preferred embodiment of the aforementioned method, PCR is performedas real-time PCR to simultaneously conduct PCR and analysis of theamplified product.

In a preferred embodiment of the aforementioned method, the analysis ofthe amplified product is performed by using a standard curverepresenting relationship between amount of the microorganism and theamplified product and created by using standard samples of themicroorganism.

As the kit of the present invention, there is provided a kit fordetecting live cells of a microorganism in a test sample bydistinguishing the live cells from dead cells or injured cells by anucleic acid amplification method, which comprises the followingcomponents:

1) an agent capable of covalently binding to a DNA or RNA by irradiationwith light having a wavelength of 350 nm to 700 nm;

2) an agent for suppressing an action of a nucleic acid amplificationinhibitory substance; and

3) a primer or primers for amplifying a target region of a DNA or RNA ofthe microorganism to be detected by a nucleic acid amplification method.

In a preferred embodiment of the aforementioned kit, the kit furthercomprises one or more kinds selected from a surfactant, a magnesiumsalt, and an organic acid salt or a phosphoric acid salt.

In a preferred embodiment of the aforementioned kit, the kit furthercomprises an enzyme having an activity of decomposing cells other thanthat of microorganism, a colloidal particle of a protein, a lipid, or asaccharide existing in the test sample.

In a preferred embodiment of the aforementioned kit, the nucleic acidamplification method is PCR, RT-PCR, LAMP, SDA, LCR, TMA, TRC, HC, orthe microarray method.

In a preferred embodiment of the aforementioned kit, the agent capableof covalently binding to a DNA or RNA by irradiation with light having awavelength of 350 nm to 700 nm is selected from ethidium monoazide,ethidium diazide, propidium monoazide, psoralen,4,5′,8-trimethylpsoralen, and 8-methoxypsoralen.

In a preferred embodiment of the aforementioned kit, the agent forsuppressing an action of a nucleic acid amplification inhibitorysubstance consists of one or more kinds selected from albumin, dextran,T4 gene 32 protein, acetamide, betaine, dimethyl sulfoxide, formamide,glycerol, polyethylene glycol, soybean trypsin inhibitor,α2-macroglobulin, tetramethylammonium chloride, lysozyme, phosphorylase,and lactate dehydrogenase.

In a preferred embodiment of the aforementioned kit, the organic acidsalt is selected from an acetic acid salt, a propionic acid salt and acitric acid salt.

In a preferred embodiment of the aforementioned kit, the phosphoric acidsalt is a pyrophosphoric acid salt.

In a preferred embodiment of the aforementioned kit, the enzyme isselected from a protease, a lipid-degrading enzyme and asaccharide-degrading enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Electrophoresis photographs of PCR amplified products obtained inthe method of the present invention “Live” represents live cells,“Injured” represents injured cells

FIG. 2 Electrophoresis photographs showing results of detection of livecells of microorganisms performed by the method of the present invention

FIG. 3 Electrophoresis photographs showing results of detection of livecells of microorganisms performed by a conventional method “Live”represents live cells, “Injured” represents injured cells

FIG. 4 Fluorescence microphotograph and stereoscopic microphotograph ofa not-heated physiological saline suspension of Enterobacter sakazakiibacterium

FIG. 5 Fluorescence microphotograph and stereoscopic microphotograph ofa supernatant of a not-heated physiological saline suspension ofEnterobacter sakazakii bacterium

FIG. 6 Fluorescence microphotograph and stereoscopic microphotograph ofa physiological saline suspension of Enterobacter sakazakii bacteriumafter repetition of thermal cycles

FIG. 7 Fluorescence microphotograph and stereoscopic microphotograph ofa supernatant of a physiological saline suspension of Enterobactersakazakii bacterium after repetition of thermal cycles

FIG. 8 Fluorescence microphotograph and stereoscopic microphotograph ofEnterobacter sakazakii bacterium suspended in a not-heated pretreatmentagent solution

FIG. 9 Fluorescence microphotograph and stereoscopic microphotograph ofa supernatant of Enterobacter sakazakii bacterium suspended in anot-heated pretreatment agent solution

FIG. 10 Fluorescence microphotograph and stereoscopic microphotograph ofEnterobacter sakazakii bacterium suspended in a pretreatment agentsolution after repetition of thermal cycles

FIG. 11 Fluorescence microphotograph and stereoscopic microphotograph ofa supernatant of Enterobacter sakazakii bacterium suspended in apretreatment agent solution after repetition of thermal cycles

FIG. 12 Drawings showing results of flow cytometry of physiologicalsaline suspensions of the Enterobacter sakazakii bacterium orsupernatants thereof in a non-heated state or after repetition ofthermal cycles

FIG. 13 Drawings showing results of flow cytometry of the Enterobactersakazakii bacterium suspended in a pretreatment agent solution orsupernatants thereof in a non-heated state or after repetition ofthermal cycles

FIG. 14 An electrophoresis photograph of the 16S-23S rRNA gene amplifiedproduct, obtained in the method of the present invention using theEnterobacter sakazakii bacterium treated with various fixationsolutions. Ct values are indicated as average and SD, and SD isindicated in parentheses

-   L: 100-bp DNA ladder-   A: Fixation solution A-   B: Fixation solution B-   C: Fixation solution C-   S: No fixation

FIG. 15 An electrophoresis photograph of the ompA gene amplifiedproduct, obtained in the method of the present invention using theEnterobacter sakazakii bacterium treated with various fixationsolutions. Ct values are indicated as average and SD, and SD isindicated in parentheses

-   A: Fixation solution A-   B: Fixation solution B-   L: 100-bp DNA ladder

FIG. 16 Electrophoresis photographs obtained before and after PCR(16S-23S rRNA gene amplification reaction) by using the Enterobactersakazakii bacterium

-   Lanes 2 and 3: supernatant of PCR reaction mixture-   Lanes 5 and 6: DNAs extracted from the pellet washed twice by the    centrifugation after PCR reaction-   Lanes 7 and 8: DNAs directly extracted from the cells-   Lanes 9 and 10: DNAs extracted from the cells actually used in the    test just before PCR-   Lanes 13 and 14: DNAs extracted from the cells washed after addition    of the PCR product-   L: 100-bp DNA ladder-   B: Fixation solution B-   S: No fixation

FIG. 17 Electrophoresis photographs of suspensions of the Enterobactersakazakii bacterium subjected to a heat treatment in the presence ofphysiological saline or pretreatment agent, and centrifugationsupernatants thereof

-   L: 100-bp DNA ladder

MODES FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of the present invention aredescribed in detail. However, the present invention is not limited tothe following preferred embodiments and may be freely modified withinthe scope of the present invention. The percentage in the presentdescription is expressed as a percentage by mass unless otherwisespecified.

A target to be detected by the method of the present invention includesall kinds of nucleic acids, specifically, single-stranded DNA,double-stranded DNA, single-stranded RNA, and double-stranded RNA, aslong as the target can be amplified eventually. Of those, the detectiontarget is preferably DNA, particularly preferably double-stranded DNA.

<1> Method of the Present Invention

The method of the present invention is a method for detecting live cellsof a microorganism in a test sample by distinguishing the live cellsfrom dead cells or injured cells, which comprises the steps of:

a) adding an agent capable of covalently binding to a DNA or RNA byirradiation with light having a wavelength of 350 nm to 700 nm to thetest sample;

b) irradiating the test sample to which the agent is added with lighthaving a wavelength of 350 nm to 700 nm;

c) amplifying a target region of the DNA or RNA of the microorganismcontained in the test sample by a nucleic acid amplification method inthe presence of an agent for suppressing an action of a nucleic acidamplification inhibitory substance, without extracting nucleic acidsfrom the cells; and

d) analyzing the amplified product.

In the specification of the present invention, the term “test sample”refers to an object containing live cells of microorganism to bedetected. The test sample is not particularly limited as long as thepresence of the microorganism can be detected by amplification of aspecific region of chromosomal DNA or RNA by a nucleic acidamplification method. Preferred examples thereof include foodstuffs,biological samples, drinking water, industrial water, environmentalwater, wastewater, soil, swab samples, and so forth.

In particular, preferred examples of the foodstuffs include: drinks suchas soft drinks, carbonated soft drinks, supplement drinks, fruit juicedrinks, and lactobacillus drinks (including concentrates and powders ofthese drinks); ice confectionery products such as ice creams, icesherbets, and shaved ice; dairy products such as processed milk, milkdrinks, fermented milk, and butter; enteral foods, fluid diets, milk forinfant, sports drinks; functional foods such as foods for specifiedhealth use and dietary supplements, and so forth.

Examples of the biological samples include blood samples, urine samples,cerebrospinal fluid samples, synovial fluid samples, pleural effusionsamples, sputum samples, feces samples, nasal cavity mucosa samples,laryngeal mucosa samples, gastric lavage solution samples, pus samples,skin mucus membrane samples, oral cavity mucosa samples, respiratoryorgan mucus membrane samples, digestive organ mucus membrane samples,eye conjunctiva samples, placenta samples, reproductive cell samples,parturient canal samples, mother's milk samples, saliva samples, vomits,contents of bulla and so forth.

Examples of the environmental water include tap water, groundwater,river water, rainwater, and so forth.

In the present invention, the test sample may be any of such foodstuffs,biological samples, drinking water, industrial water, environmentalwater, wastewater, soil, swab samples as mentioned above, etc.themselves or may be any of diluted or concentrated products thereof orany of products obtained by a pretreatment other than the method of thepresent invention. Examples of the pretreatment include heat treatment,filtration, centrifugation, and so forth.

Further, foreign substances such as cells other than microorganisms,protein colloidal particles, lipids, saccharides and so forth in thetest sample may be removed or reduced by a treatment with an enzymehaving an activity for degrading them or the like. In the case where thetest sample is any of milk, dairy products, and foods produced from milkor dairy products, examples of the cells other than microorganisms inthe test sample include bovine leukocytes, mammary epitheliocytes, andso forth. Meanwhile, in the case where the test sample is any ofbiological samples such as blood samples, urine samples, spinal fluidsamples, synovial fluid samples, and pleural effusion samples, examplesof the cells include erythrocytes, leukocytes (such as granulocytes,neutrophils, basophils, monocytes, and lymphocytes), thrombocytes, andso forth.

The enzyme is not particularly limited as long as it can degrade theforeign substances and does not damage live cells of microorganism to bedetected, and examples thereof include, for example, lipid-degradingenzymes, protein-degrading enzymes and saccharide-degrading enzymes. Ofthose, one enzyme or two or more enzymes may be used, but it ispreferable to use both the lipid-degrading enzyme and protein-degradingenzyme, or all of the lipid-degrading enzyme, protein-degrading enzymeand saccharide-degrading enzyme.

Examples of the lipid-degrading enzymes include lipase, phosphatase, andso forth, examples of the protein-degrading enzymes include serineprotease, cysteine protease, proteinase K, pronase, and so forth, andexamples of the saccharide-degrading enzyme include amylase, cellulase,and so forth.

The “microorganism” is an object to be detected by the method of thepresent invention, and is not particularly limited so long as it can bedetected by nucleic acid amplification methods, and the agent capable ofcovalently binding to a DNA or RNA by irradiation with light having awavelength of 350 nm to 700 nm act on live cells of the microorganism ina manner different from that for dead cells and injured cells of themicroorganism. Preferred examples include bacteria, fungi, yeasts,viruses, and so forth. The bacteria include both gram-positive bacteriaand gram-negative bacteria. Examples of the gram-positive bacteriainclude Staphylococcus bacteria such as Staphylococcus epidermidis,Streptococcus bacteria such as Streptococcus pneumoniae, Listeriabacteria such as Listeria monocytogenes, Bacillus bacteria such asBacillus cereus and Bacillus anthracis, Mycobacterium bacteria such asMycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium,Clostridium bacteria such as Clostridium botulinum and Clostridiumperfringens and so forth. Examples of the gram-negative bacteria includeenterobacteria of which typical examples are Escherichia bacteria suchas Escherichia coli, Enterobacter bacteria such as Enterobactersakazakii, Citrobacter bacteria such as Citrobacter koseri, andKlebsiella bacteria such as Klebsiella oxytoca, and, Salmonellabacteria, Vibrio bacteria, Pseudomonas bacteria, Legionella bacteria,and so forth. Examples of virus include viruses having an envelope suchas influenza virus, and viruses not having envelope but having only anucleocapsid, such as noroviruses, rotaviruses and adenoviruses.

As for viruses, there is known a method for measuring activation andinactivation of virus in water, in which a photoreactive nucleic acidcross-linker (EMA) is allowed to act on a test sample, and then onlyactivated viruses are measured by RT-PCR (Development of ethidiummonoazide (EMA)-RT-PCR for selective detection of enteric viruses, 15thInternational Symposium on Health-Related Water Microbiology, (May31-Jun. 5, 2009, Ursulines Conference Centre, Naxos, Greece)). That is,it is suggested that EMA does not penetrate activated viruses, butpenetrates only inactivated viruses having severely physically injurednucleocapsids, and therefore, activated viruses (live) and inactivatedviruses (dead) can be distinguished with EMA. Therefore, it is thoughtthat the present invention can be applied not only to bacteria,filamentous fungi and yeast, but also to viruses.

In the present invention, the “live cell” refers to a cell in a statethat the cell can proliferate, and exhibits metabolic activities of themicroorganism (viable-and-culturable state), when it is cultured undergenerally preferred culture conditions, and is a cell substantially freefrom injury of cell wall. As the metabolic activities mentioned above,ATP activity, esterase activity etc. can be exemplified. In the presentinvention, viral particles are also called “cells” for convenience. Asfor viruses, “live cell” refers to a virus in a state that it can infecta mammalian cell and proliferate.

The “dead cell” is a cell in a state that it cannot proliferate, anddoes not exhibit metabolic activities (dead state), even if it iscultured under an optimum culture condition. Moreover, it is in a statethat although structure of cell wall is maintained, the cell wall itselfis highly injured, and a nuclear stain agent exhibiting weakpermeability such as propidium iodide can penetrate or permeate the cellwall. As for viruses, “dead cell” refers to a virus in a state that itcannot infect a mammalian cell.

The “injured cell” (injured cell or viable-but-non culturable cell) is acell in a state that even when it is cultured under a generallypreferred culture condition, it hardly proliferates because it isinjured due to artificial stress or environmental stress, and it showsmetabolic activities at a lower level compared with a live cell, but asignificant level compared with a dead cell. As for viruses, “injuredcell” refers to a virus in a state that even if it infects a mammaliancell, it cannot proliferate in the cell.

In this specification, unless specially mentioned, the “live cell”,“dead cell”, and “injured cell” mean a live cell, dead cell, and injuredcell of a microorganism, respectively.

Detection of bacteria exhibiting the state of injured cells due to mildheat treatment or administration of antibiotics is attracting attention,in particular, in the field of food sanitation inspection and clinicaltest, and the present invention provides a method for detecting amicroorganism, which enables not only detection of live cells, but alsodistinction of live cells from dead cells or injured cells.

The unit of cell number is usually cell number (cells)/ml for all oflive cells, injured cells and dead cells. In this specification, thecell number is represented by a logarithm, and “a log₁₀ cells/ml” means10^(a) cells/ml.

The number of live cells can be approximated with a number of formedcolonies (cfu/ml (colony forming units/ml)) obtainable by culturing thecells under an optimum condition on a suitable plate medium. A standardsample of injured cells of microorganism can be prepared by, forexample, subjecting a live cell suspension to a heat treatment, forexample, a heat treatment in boiling water. The number of injured cellsin such a sample can be approximated with cfu/ml in the live cellsuspension before the heat treatment. Although time of the heattreatment in boiling water for preparing injured cells varies dependingon type of microorganism, injured cells of the bacteria described in theexamples, for example, can be prepared by a heat treatment of about 50seconds. Further, a standard sample of injured cells of microorganismcan also be prepared by a treatment with an antibiotic. In such a case,the cell number of the injured cells can be approximated based on thenumber of formed colonies (cfu/ml) observed when the cells are culturedunder an optimum condition on a suitable plate medium, that is, a livecell suspension is treated with an antibiotic, then the antibiotic isremoved, transmittance of visible light (wavelength: 600 nm) through thesuspension, i.e., turbidity of the suspension, is measured, and themeasured turbidity can be compared with that of a live cell suspensionof a known live cell density to calculate the number of injured cellstreated with the antibiotic.

As for viruses, unit of cell number is represented by plaque-formingunit (pfu or PFU).

The method of the present invention is for detection of live cells ofmicroorganism, and cells of the microorganism distinguished from livecells may be injured cells or dead cells.

In the present invention, the “detection of live cells” includes bothdetermination of presence or absence of live cells in a test sample anddetermination of amount of live cells in a test sample. The amount oflive cells is not limited to an absolute amount, and may be a relativeamount with respect to that in a control sample. Moreover, “to detectlive cells by distinguishing the live cells from dead cells or injuredcells” means to more selectively detect live cells compared with deadcells or injured cells. In addition, the “distinction of live cells fromdead cells or injured cells” includes distinction of live cells fromboth dead cells and injured cells.

Hereafter, the method of the present invention will be explained foreach step. As described above, the method of the present invention maycomprise the step of treating the test sample with an enzyme having anactivity of decomposing cells other than those of microorganism,colloidal particles of proteins, lipids, or saccharides existing in thetest sample, before the steps described below.

(1) Step a)

An agent capable of covalently binding to a DNA or RNA by irradiationwith light having a wavelength of 350 nm to 700 nm is added to the testsample. That is, microorganisms in the test sample are treated with theagent.

As described later, the agent intercalates into a double-stranded DNA orRNA and covalently binds to the DNA or RNA by irradiation with light tocross-link the molecules. Further, it is estimated that the agentcovalently binds to a single-stranded DNA or RNA by irradiation withlight to inhibit reactions of PCR. The agent may henceforth also besimply referred to as “cross-linker”.

The cross-linker preferably has different actions on live cells ofmicroorganism compared with injured or dead cells of microorganism andsomatic cells such as bovine leukocytes, leukocytes, or thrombocytes.More specifically, the cross-linker is preferably one which can moreeasily pass through cell walls of injured or dead cells of microorganismand cell membranes of somatic cells such as bovine leukocytes,leukocytes, or thrombocytes compared with cell walls of live cells ofmicroorganism.

Examples of the cross-linker include ethidium monoazide, ethidiumdiazide, psoralen, 4,5′,8-trimethylpsoralen, 8-methoxypsoralen,propidium monoazide, and so forth. One kind of those cross-linkers maybe used alone, or two or more kinds thereof may be used in combination.

The conditions of the treatment with the cross-linker may beappropriately set. For example, conditions for easily distinguishinglive cells of microorganism from dead or injured cells of microorganismcan be determined by: adding various concentrations of a cross-linker tosuspensions of live cells of microorganism to be detected and dead orinjured cells of microorganism; allowing the suspensions to stand forvarious periods of time; separating the cells by centrifugation; andanalyzing the cells by the nucleic acid amplification method. Moreover,conditions for easily distinguishing live cells of microorganism fromvarious cells can be determined by: adding various concentrations of across-linker to suspensions of live cells of microorganism to bedetected and somatic cells such as bovine leukocytes or thrombocytes;allowing the suspensions to stand for various periods of time;separating the cells and the various cells by centrifugation; andanalyzing the cells by the nucleic acid amplification method.Specifically: conditions of a treatment with ethidium monoazide at afinal concentration of 1 to 100 μg/ml for 5 minutes to 48 hours at 4 to10° C.; conditions of a treatment with ethidium diazide at a finalconcentration of 1 to 100 μg/ml for 5 minutes to 48 hours at 4 to 10°C.; conditions of a treatment with propidium monoazide at a finalconcentration of 1 to 100 μg/ml for 5 minutes to 48 hours at 4 to 10°C.; conditions of a treatment with psoralen at a final concentration of1×10⁻⁵ to 10 μg/ml for 5 minutes to 48 hours at 25 to 37° C.; conditionsof a treatment with 4,5′,8-trimethylpsoralen at a final concentration of1×10⁻⁵ to 10 μg/ml for 5 minutes to 48 hours at 25 to 37° C.; andconditions of a treatment with 8-methoxypsoralen at a finalconcentration of 1×10⁻⁵ to 10 μg/ml for 5 minutes to 48 hours at 25 to37° C. are exemplified.

(2) Step b)

Next, each test sample containing the cross-linker is irradiated withlight having a wavelength of 350 nm to 700 nm.

The cross-linker can easily pass through cell walls of dead cells andinjured cells compared with cell walls of live cells of microorganism.Therefore, it is presumed that the cross-linker does not substantiallypass through cell walls of live cells of microorganism but passesthrough cell walls of injured or dead cells of microorganism, or deadsomatic cells, provided that the treatment is performed within theabove-mentioned periods of time. It is presumed that, as a result, thecross-linker moves into dead somatic cells, dead and injured cells ofmicroorganism, and subsequently form hydrogen bonds with chromosomal DNAor RNA, and then the cross-linker irradiated with light having awavelength of 350 nm to 700 nm cross-links DNA molecules or covalentlybinds with RNA and, as a result, causes a deformation in the chromosomalDNA or modification of RNA with the cross-linker, eventually resultingin disruption (fragmentation/cleavage) of the chromosomal DNA, or RNAnot serving as a template of a nucleic acid amplification reaction.

The light having a wavelength of 350 nm to 700 nm may include at leastlight having a wavelength of 350 nm to 700 nm, and the light may besingle-wavelength light or multi-wavelength light. In addition, alllight components may be in the range of 350 nm to 700 nm, or the lightmay include short-wavelength light having a wavelength shorter than 350nm and/or long-wavelength light having a wavelength longer than 700 nm.The peak in an intensity distribution is preferably in the range of 350nm to 700 nm. Preferably, the light does not include a component with ashort wavelength to such a degree that chromosomal DNA of amicroorganism is cleaved only by light irradiation.

When chromosomal DNA of injured or dead cells is more preferentiallydisrupted than that of live cells of microorganism, a target region ofthe chromosomal DNA of the live cells is amplified by the nucleic acidamplification method, while a target region of the injured or dead cellsis disrupted (cleaved), resulting in inhibiting the nucleic acidamplification reactions. As a result, the live cells of microorganismcan be detected more selectively than the injured or dead cells.

Further, when RNA of injured or dead cells is more preferentiallymodified with the cross-linker than that of live cells of microorganism,a target region of RNA of the live cells is amplified by the nucleicacid amplification method, while a target region of RNA of the injuredor dead cells is modified, resulting in inhibiting the nucleic acidamplification reactions. As a result, the live cells of microorganismcan be detected more selectively than the injured or dead cells.

In a preferred embodiment of the present invention, the cross-linker isethidium monoazide, and the method includes the step of irradiating atest sample to which ethidium monoazide is added with light having awavelength of 350 nm to 700 nm. Ethidium monoazide (EMA) can easily passthrough cell walls of injured or dead cells compared with cell walls oflive cells of microorganism. Therefore, it is presumed that EMA does notsubstantially pass through cell walls of live cells of microorganism butpasses through cell walls of injured or dead cells of microorganism orcell membranes of dead somatic cells.

Note that, in the case where leukocytes and thrombocytes in blood arelive cells, EMA can more easily pass through cell walls of the cells insterilized water or a hypotonic salt solution.

Concerning DNA, in particular, EMA moves into dead somatic cells andinjured and dead cells of microorganism and intercalates randomly intonuclear DNA, and intercalated EMA is converted into nitrene byirradiation with light having a wavelength of 350 nm to 700 nm and bindscovalently to nuclear DNA to cross-link DNA molecules. Then, it ispresumed that EMA which binds covalently to bases and deoxyriboses inchromosomal DNA at many points causes a large deformation in thechromosomal DNA, resulting in disrupting (fragmentating) the chromosomalDNA.

Further, concerning double-stranded RNA (including partiallydouble-stranded RNA), EMA moves into dead somatic cells and injured anddead cells of microorganism and intercalates randomly into RNA, and thenintercalated EMA is converted into nitrene by irradiation with lighthaving a wavelength of 350 nm to 700 nm and binds covalently to the RNAto cross-link RNA molecules. Then, it is presumed that EMA which bindscovalently to bases of RNA at many points causes a large deformation inthe RNA, resulting in disrupting (fragmentating) the RNA.

Furthermore, concerning single-stranded DNA or RNA, it is presumed thatEMA moves into dead somatic cells and injured and dead cells ofmicroorganism, and then converted into nitrene by irradiation with lighthaving a wavelength of 350 nm to 700 nm, which binds covalently to theDNA or RNA.

A cross-linker other than ethidium monoazide may also be used so long asthe cross-linker can more easily pass through cell walls of injured ordead cells compared with cell walls of live cells of microorganism andcan cross-link DNA or covalently binds to RNA by irradiation with lighthaving a wavelength of 350 nm to 700 nm (long-wavelength ultravioletlight or visible light) to thereby disrupt the chromosomal DNA or modifyRNA.

Conditions for the treatment with EMA can be appropriately determined.For example, conditions that enables easy distinction of live cells ofmicroorganism from dead cells and injured cells can be determined byadding EMA at various concentrations to suspensions of live cells andinjured cells or dead cells of the microorganism to be detected, leavingthem for various periods of time, then irradiating them with visiblelight, removing the cells by centrifugation or the like as required, andperforming analysis by nucleic acid amplification methods. Preferredconditions for the irradiation of light can also be appropriatelydetermined by performing such an experiment as mentioned above usingvarious irradiation times. Specific examples of the conditions for theirradiation of light include irradiation of lights of 100 to 750 W andthe aforementioned wavelength for 5 minutes to 2 hours from a distanceof 10 to 50 cm from the test sample. The irradiation of light ispreferably performed at a low temperature, for example, with ice coolingof the sample.

The addition of the cross-linker in the aforementioned step a) and thelight irradiation treatment of the step b) may be repeated for 2 or morecycles. In such a case, the concentration of the cross-linker ispreferably made higher in the step a) of the first time compared withthat in the step a) of the second time or thereafter, and made lower inthe step a) of the second time or thereafter compared with that in thestep a) of the first time.

For example, if EMA is allowed to act at a high concentration, forexample, 10 μg/ml or higher, although permeability for the cell wall orcell membrane of dead cells becomes higher, the permeability for livecells also becomes high (Microbiology and Immunology, 2007, 51, pp.763-775; Journal of Clinical Microbiology, 2008, 46, pp. 2305-2313). Onthe other hand, if EMA is allowed to act at a low concentration, forexample, lower than 10 μg/ml, although permeation into live cells can beavoided, permeation rate into dead cells also decreases, and therefore,dead cells may also be detected by nucleic acid amplification reactions.Therefore, it is preferable to use a high concentration of thecross-linker in the step a) of the first time, and make theconcentration of the cross-linker low in the step b) of second time andthereafter.

Specifically, the final concentration of the cross-linker in the step a)of the first time is, for example, 10 to 100 μg/ml in the case ofethidium monoazide, 10 to 100 μg/ml in the case of ethidium diazide, 10to 100 μg/ml in the case of propidium monoazide, 2×10⁻⁵ to 10 μg/ml inthe case of psoralen, 2×10⁻⁵ to 10 μg/ml in the case of4,5′,8-trimethylpsoralen, or 2×10⁻⁵ to 10 μg/ml in the case of8-methoxypsoralen. Further, the final concentration of the cross-linkerin the step a) of the second time and thereafter is, for example, 1 to10 μg/ml in the case of ethidium monoazide, 1 to 10 μg/ml in the case ofethidium diazide, 1 to 10 μg/ml in the case of propidium monoazide,1×10⁻⁵ to 9 μg/ml in the case of psoralen, 1×10⁻⁵ to 9 μg/ml in the caseof 4,5′,8-trimethylpsoralen, or 1×10⁻⁵ to 9 μg/ml in the case of8-methoxypsoralen.

Further, the treatment time of the step a) of the first time ispreferably made shorter than that of the step a) of the second time andthereafter. Specifically, the treatment time of the step a) of the firsttime is, for example, 5 minutes to 1 hour in the case of ethidiummonoazide, 5 minutes to 1 hour in the case of ethidium diazide, 5minutes to 1 hour in the case of propidium monoazide, 5 minutes to 1hour in the case of psoralen, 5 minutes to 1 hour in the case of4,5′,8-trimethylpsoralen, or 5 minutes to 1 hour in the case of8-methoxypsoralen. The treatment time of the step a) of the second timeand thereafter is, for example, 6 minutes to 48 hours in the case ofethidium monoazide, 6 minutes to 48 hours in the case of ethidiumdiazide, 6 minutes to 48 hours in the case of propidium monoazide, 6minutes to 48 hours in the case of psoralen, 6 minutes to 48 hours inthe case of 4,5′,8-trimethylpsoralen, or 6 minutes to 48 hours in thecase of 8-methoxypsoralen.

Between the step b) of a previous cycle and the step a) of a followingcycle, the step of removing the unreacted cross-linker may be added.Moreover, between the step b) and the step c) described below, the stepof removing the cross-linker may be added. The cross-linker that doesnot react in the step a) is usually substantially inactivated in thestep b). Therefore, as a method for removing the cross-linker, a methodof centrifuging the test sample to separate precipitates containingmicroorganism and a supernatant containing the cross-linker, andremoving the supernatant is mentioned. In this case, after thecross-linker is removed, the step of washing the microorganism with awashing agent may optionally be added.

(3) Step c)

Then, a target region of the DNA or RNA of the microorganism containedin the test sample after the light irradiation treatment is amplified bya nucleic acid amplification method in the presence of an agent forsuppressing an action of a nucleic acid amplification inhibitorysubstance, without extracting nucleic acids from the cells.

Specifically, the agent for suppressing an action of a nucleic acidamplification inhibitory substance is added to a nucleic acidamplification reaction solution containing the test sample, and thenucleic acid amplification reaction is performed.

Furthermore, in addition to the agent for suppressing an action of anucleic acid amplification inhibitory substance, a surfactant, amagnesium salt, or an organic acid salt or a phosphoric acid salt may beadded to the amplification reaction solution. These substances may beused independently or as a combination of any two or more kinds ofthese. It is particularly preferable to add all of these substances. Theorder of the addition of the agent for suppressing an action of anucleic acid amplification inhibitory substance, the surfactant, themagnesium salt, and the organic acid salt or the phosphoric acid salt isnot limited, and they may be simultaneously added.

The nucleic acid amplification inhibitory substance is a substance whichinhibits a nucleic acid amplification reaction or a nucleic acidextension reaction, and examples include positively charged inhibitorysubstances adsorbing to template of nucleic acid (DNA or RNA),negatively charged inhibitory substances adsorbing to nucleic acidbiosynthesis enzymes (DNA polymerase etc.), and so forth. Examples ofthe positively charged inhibitory substances include calcium ion,polyamines, heme, and so forth. Examples of the negatively chargedinhibitory substances include phenol, phenol type compounds, heparin,gram-negative bacterium cell wall having outer membranes, and so forth.It is said that such substances that inhibit a nucleic acidamplification reaction are abundantly contained in foodstuffs orclinical test samples.

Examples of the agent for suppressing an action of a nucleic acidamplification inhibitory substance mentioned above include one or morekinds selected from albumin, dextran, T4 gene 32 protein, acetamide,betaine, dimethyl sulfoxide, formamide, glycerol, polyethylene glycol,soybean trypsin inhibitor, α2-macroglobulin, tetramethylammoniumchloride, lysozyme, phosphorylase, and lactate dehydrogenase. Examplesof the polyethylene glycol include polyethylene glycol 400 andpolyethylene glycol 4000. Examples of the betaine includetrimethylglycine, derivatives thereof, and so forth. Examples of thephosphorylase and lactate dehydrogenase include rabbit muscular glycogenphosphorylase and lactate dehydrogenase. As the glycogen phosphorylase,glycogen phosphorylase b is preferred.

It is particularly preferable to use albumin, dextran, T4 gene 32protein, or lysozyme.

As an attempt to reduce the inhibitory action of the nucleic acidamplification inhibitory substance contained in a test sample for whichblood, feces, and meat are supposed, reduction of the inhibitory actionby adding such substances as described above to a PCR reaction mixtureis evaluated (Abu Al-Soud, W. et al, Journal of Clinical Microbiology,38:4463-4470, 2000).

There is suggested a possibility that albumin of which representativeexample is BSA (bovine serum albumin) may reduce inhibition of nucleicacid amplification by binding to a nucleic acid amplification inhibitorysubstance such as heme (Abu Al-Soud et al., as mentioned above).Moreover, there are considered two ways of possibility, that is, the T4gene 32 protein is a single-stranded DNA binding protein, and it bindsto a single-stranded DNA serving as a template in a nucleic acidamplification process in advance so that degradation of the template bya nuclease is avoided, and the nucleic acid amplification reaction isnot inhibited, but promoted, or it binds to a nucleic acid amplificationinhibitory substance like BSA so that the nucleic acid amplificationreaction is not inhibited, but promoted (Abu Al-Soud et al., asmentioned above). Furthermore, there is further suggested a possibilitythat BSA, the T4 gene 32 protein, and a proteinase inhibitor bind to aproteinase to reduce the proteolysis activity to bring out the actionsof nucleic acid biosynthesis enzymes to the maximum extent. In fact,proteinases may remain in cow's milk or blood, and an example is alsoreported that, in such a case, decomposition of nucleic acidbiosynthesis enzymes was avoided by adding BSA or a proteinase inhibitor(e.g., soybean trypsin inhibitor and α2-macroglobulin), and the nucleicacid amplification reaction favorably advanced (Abu Al-Soud et al., asmentioned above). Further, dextran is generally a polysaccharide that issynthesized by lactic acid bacteria using glucose as a raw material, andit is also reported that a complex of a similar polysaccharide andpeptide called mucin adheres to the intestinal mucosa (Ruas-Madiedo, P.,Applied and Environmental Microbiology, 74:1936-1940, 2008). Therefore,it is possibly estimated that dextran adsorbs to negatively chargedinhibitory substances (adsorbs to nucleic acid biosynthesis enzymes), orpositively charged inhibitory substances (adsorbs to nucleic acids) inadvance, and then binds to these inhibitory substances.

Moreover, it is estimated that lysozyme adsorbs to nucleic acidamplification inhibitory substances considered to be abundantlycontained in cow's milk (Abu Al-Soud et al., as mentioned above).

From the above, it can be said that such substances as mentioned aboverepresented by albumin, T4 gene 32 protein, dextran, and lysozyme areagents for suppressing an action of a nucleic acid amplificationinhibitory substance.

Examples of albumin include bovine serum albumin, ovalbumin,lactalbumin, human serum albumin, and so forth. Among these, bovineserum albumin is preferred. Albumin may be a purified product, andunless the effect of the present invention is degraded, it may containother components such as globulin. Further, albumin may also be afractionation product. Concentration of albumin in the test sample(nucleic acid amplification reaction solution) is, for example, usually0.0001 to 1 mass %, preferably 0.01 to 1 mass %, more preferably 0.2 to0.6 mass %.

Examples of dextran include dextran 40, dextran 500, and so forth. Amongthese, dextran 40 is preferred. Concentration of the dextran in the testsample (nucleic acid amplification reaction solution) is, for example,usually 1 to 8%, preferably 1 to 6%, more preferably 1 to 4%.

Concentration of the T4 gene 32 protein (e.g., produced by Roche A. G.,also called 32) in the test sample (nucleic acid amplification reactionsolution) is usually 0.01 to 1%, preferably 0.01 to 0.1%, morepreferably 0.01 to 0.02%.

Examples of lysozyme include lysozyme derived from egg white.Concentration of lysozyme in the test sample (nucleic acid amplificationreaction solution) is, for example, usually 1 to 20 μg/ml, preferably 6to 15 μg/ml, more preferably 9 to 13 μg/ml.

Examples of the surfactant include nonionic surfactants such as those ofTriton (registered trademark of Union Carbide), Nonidet (Shell), Tween(registered trademark of ICI) and Brij (registered trademark of ICI)series, anionic surfactants such as SDS (sodium dodecylsulfate), andcationic surfactants such as stearyldimethylbenzylammonium chloride.Examples of the Triton series surfactant include Triton X-100 etc.,examples of the Nonidet series surfactant include Nonidet P-40 etc.,examples of the Tween series surfactant include Tween 20, Tween 40,Tween 60, Tween 80 etc., and examples of the Brij series surfactantinclude Brij 56 etc.

Type and concentration of the surfactant in the nucleic acidamplification reaction solution are not particularly limited, so long asthe penetration of the PCR reagents into cells of the microorganism ispromoted, and the nucleic acid amplification reaction is notsubstantially inhibited. Specifically, concentration of SDS is, forexample, usually 0.0005 to 0.01%, preferably 0.001 to 0.01%, morepreferably 0.001 to 0.005%, still more preferably 0.001 to 0.002%. Asfor the other surfactants, for example, in the case of Nonidet P-40, theconcentration is usually 0.001 to 1.5%, preferably 0.002 to 1.2%, morepreferably 0.9 to 1.1%. In the case of Tween 20, the concentration isusually 0.001 to 1.5%, preferably 0.002 to 1.2%, more preferably 0.9 to1.1%. In the case of Brij56, the concentration is usually 0.1 to 1.5%,preferably 0.4 to 1.2%, more preferably 0.7 to 1.1%.

When a surfactant is contained in an enzyme solution used for thenucleic acid amplification reaction, the surfactant may consist of onlythe surfactant derived from the enzyme solution, or a surfactant of thesame type or different type may be further added.

Examples of the magnesium salt include magnesium chloride, magnesiumsulfate, magnesium carbonate, and so forth. Concentration of themagnesium salt in the test sample (nucleic acid amplification reactionsolution) is, for example, usually 1 to 10 mM, preferably 2 to 6 mM,more preferably 2 to 5 mM.

Examples of the organic acid salt include salts of citric acid, tartaricacid, propionic acid, butyric acid, and so forth. Examples of type ofthe salt include sodium salt, potassium salt, and so forth. Further,examples of the phosphoric acid salt include salts of pyrophosphoricacid and so forth. These may be used independently or as a mixture oftwo or three or more kinds of them. Concentration of the organic acidsalt or the phosphoric acid salt in the test sample (nucleic acidamplification reaction solution) is, for example, usually 0.1 to 20 mM,preferably 1 to 10 mM, more preferably 1 to 5 mM, in terms of the totalamount.

In the present invention, extraction of nucleic acids from the cells isnot performed, which is performed before the nucleic acid amplificationreaction in conventional methods. The extraction of nucleic acids fromcells means, for example, collection or purification of nucleic acidsfrom cells disrupted or lysed by an enzymatic or physical means. In thepresent invention, such a treatment for extracting nucleic acids fromcells, for example, a treatment of collecting or purifying nucleic acidsby disrupting or lysing the cells by an enzymatic or physical means, isnot performed.

A target region of the DNA or RNA which has existed in the cells isamplified by a nucleic acid amplification method in the presence of theagent for suppressing an action of a nucleic acid amplificationinhibitory substance, and other components, if needed. As the templatefor the nucleic acid amplification, a microbial cell suspension or amicrobial cell suspension treated with a protein-degrading enzyme, alipid-degrading enzyme, a saccharide-degrading enzyme etc. is used, andextraction of nucleic acids for preparing the template is not performed.The nucleic acid amplification method preferably comprises the step ofthermal denaturation of nucleic acids at a high temperature, forexample, 90 to 95° C., preferably 93 to 95° C., more preferably 94 to95° C.

The amplification of the target region is preferably performed inmicrobial cells. In the present invention, the amplification is highlypossibly attained in microbial cells as shown in the examples. That is,it is presumed that the morphology of the cells is maintained by thehigh temperature treatment in the nucleic acid amplification reaction,and, in a preferred embodiment, actions the of aforementionedcomponents, so that the chromosomal DNA is retained in the cells, butpinholes or voids are formed in the cell membranes or cell walls of themicroorganism, thus primers, enzymes required for the nucleic acidamplification etc. flow into the cells, the amplification reactionoccurs in the cells, and then a part of the amplified product remains inthe cells or flow out of the cells depending on the gene length of theamplified product. However, a possibility that an extremely small partof the chromosomal DNA or RNA flows out of the cells through thepinholes or voids of the cell membranes or cell walls cannot be denied,either.

In any case, inflow of components required for the nucleic acidamplification such as primers into the cells without substantialdisruption or lysis of the cells, retention in the cells or outflow fromthe cells of a part of the amplified product, and outflow of thechromosomal DNA or RNA from the cells are not included in the“extraction of nucleic acids”. Moreover, although existence of amechanism other than that described above cannot be negated, even insuch a case, the method complies with the definition that “extraction ofnucleic acids is not performed”, so long as a treatment of collecting orpurifying nucleic acids by disrupting or lysing the cells by, forexample, an enzymatic or physical means, is not performed.

In addition, even when the chromosomal DNA or RNA flown out from thecells serves as a template, and the nucleic acid amplification reactionoccurs out of the cells, if the major amplified product is formed in thecells, it can be said that the nucleic acid amplification reaction is“performed in microbial cells”. Specifically, for example, if 80% ormore, preferably 90% or more, more preferably 99% or more, of theamplified product is formed in the microbial cells, it can be estimatedthat the nucleic acid amplification reaction is performed in themicrobial cells.

Examples of the nucleic acid amplification method include PCR method(White, T. J. et al., Trends Genet., 5, 185 (1989)), LAMP method(Loop-Mediated Isothermal Amplification: Principal and application ofnovel gene amplification method (LAMP method), Tsugunori Notomi, ToruNagatani, BIO INDUSTRY, Vol. 18, No. 2, 15-23, 2001), SDA method (StrandDisplacement Amplification: Edward L. Chan, et al., Arch. Pathol. Lab.Med., 124:1649-1652, 2000), LCR method (Ligase Chain Reaction: Barany,F., Proc. Natl. Acad. Sci. USA, Vol. 88, p. 189-193, 1991), TMA method(Transcription-Mediated-Amplification: Sarrazin C. et al., J. Clin.Microbiol., vol. 39: pp. 2850-2855 (2001)), TRC method(Transcription-Reverse Transcription-Concerted method: Nakaguchi Y. etal., J. Clin. Microbiol., vol. 42: pp. 4248-4292 (2004)), HC method(Hybrid Capture: Nazarenko I., Kobayashi L. et al., J. Virol. Methods,vol. 154: pp. 76-81, 2008), microarray method (Richard P. Spence, etal., J. Clin. Microbiol., Vol. 46, No. 5, pp. 1620-1627, 2008), and soforth. In the present invention, although the PCR method is particularlypreferably used, the nucleic acid amplification method is not limitedthereto.

In the present invention, the “target region” is not particularlylimited so long as a region of a chromosomal DNA or RNA that can beamplified by PCR using primers used for the present invention andenables detection of a microorganism to be detected is selected, and itcan be suitably selected depending on the purpose. For example, whencells of a type different from that of the microorganism to be detectedare contained in the test sample, the target region preferably containsa sequence specific to the microorganism to be detected. Further,depending on the purpose, the target region may be one containing asequence common to several kinds of microorganisms. Furthermore, thetarget region may consist of a single region or two or more regions. Ifa primer set suitable for a target region specific to the microorganismto be detected and a primer set suitable for chromosomal DNAs of widevarieties of microorganisms are used, live cell amount of themicroorganism as the object of the detection and live cell amount of thewide varieties of microorganisms can be simultaneously measured. Lengthof the target region is, for example, usually 50 to 5000 nucleotides.

The primers to be used in amplification of a nucleic acid may beselected based on principles of various nucleic acid amplificationmethods and are not particularly limited as long as the primers canspecifically amplify the above-mentioned target region.

Preferred examples of the target region include various specific genessuch as a 5S rRNA gene, 16S rRNA gene, 23S rRNA gene, tRNA gene, andpathogen gene. Any one of these genes or a part of any one of thesegenes may be targeted, or a region extending over two or more genes maybe targeted. For example, as for coliform bacteria and bacteria of thefamily Enterobacteriaceae, a part of the 16S rRNA gene can be amplifiedby using the primer set shown in SEQ ID NOS: 1 and 2. Further, a regionextending over a part of the 16S rRNA gene, a tRNA gene, and a part ofthe 23S rRNA gene can be amplified by using the primer set shown in SEQID NOS: 3 and 4.

In the case where the microorganism of the detection target is apathogenic bacterium, the target region may be a pathogenic gene.Examples of the pathogenic gene include: listeriolysin O (hlyA) gene ofa Listeria bacterium; enterotoxin gene and invasion (invA) gene of aSalmonella bacterium; verotoxin genes of pathogenic Escherichia coliO-157, O-26, O-111 etc.; outer-membrane-protein A (ompA) gene(Enterobacter sakazakii) and macromolecular synthesis (MMS) operon(Enterobacter sakazakii) of an Enterobacter bacterium;macrophage-invasion protein (mip) gene of a Legionella bacterium;heat-resistant hemolysin gene and heat-resistant hemolysin-like toxingene of Vibrio parahaemolyticus; ipa gene (invasion plasmid antigengene) and invE gene (invasion gene) of Shigella dysenteriae andenteroinvasive Escherichia coli; enterotoxin gene of Staphylococcusaureus; cereulide gene and enterotoxin gene of Bacillus cereus; varioustoxin genes of Clostridium botulinum, and so forth. Further, examples ofprimers for a pathogenic gene include, for example, a set of primersshown in SEQ ID NOS: 5 and 6 for hlyA gene of Listeria bacterium; a setof primers shown in SEQ ID NOS: 7 and 8 for ompA gene of Enterobactersakazakii; and a set of primers shown in SEQ ID NOS: 9 and 10 for MMSoperon of Enterobacter sakazakii.

Furthermore, in the case of the influenza virus having an envelope,examples of the target region include the hemagglutinin (H protein) geneand the neuraminidase (N protein) gene, RNA polymerase gene of theCalicivirus family viruses of which representative examples includenoroviruses, genetic regions coding for various capsid proteins, and soforth. As food poisoning viruses, besides noroviruses, there are alsorotaviruses and adenoviruses, and as the objective gene, RNA polymerasegenes and genetic regions coding for various capsid proteins thereof maybe used as the target region as in the case of noroviruses.

If primers suitable for two or more kinds of microorganisms are used,live cells of two or more kinds of the microorganisms in a test samplecan be detected. Moreover, if a primer(s) specific to a particularbacterium are used, live cells of the particular bacterium in a testsample can be detected.

Conditions of nucleic acid amplification reactions are not particularlylimited as long as a nucleic acid can be specifically amplified based onprinciples of various nucleic acid amplification methods (such as PCR,LAMP, SDA, LCR, TMA, TRC, HC, microarray method, etc.), and theconditions may be appropriately set.

(4) Step d)

Subsequently, amplified products obtained by the nucleic acidamplification method are analyzed. The analysis of the amplifiedproducts is performed following the step c), or performed simultaneouslywith the step c), depending on the nucleic acid amplification methodadopted in the step c). For example, in the case of using real-time PCR,the step d) may be performed simultaneously with the step c).

The analysis method is not particularly limited as long as the methodcan detect or quantify the nucleic acid amplified products, and examplesthereof include electrophoresis. Note that, in the case of using PCRmethod for the nucleic acid amplification method, a real-time PCR method(Nogva et al., Appl. Environ. Microbiol., vol. 66, 2000, pp. 4266-4271;Nogva et al., Appl. Environ. Microbiol., vol. 66, 2000, pp. 4029-4036)can be used.

The electrophoresis enables evaluation of the amounts and sizes of thenucleic acid amplified products. In addition, real-time PCR enablesrapid quantification of PCR amplified products.

In the case where the real-time PCR is employed, changes in fluorescentintensities are generally noise levels and about zero if the number ofamplification cycles is in the range of 1 to 10. Therefore, theintensities are regarded as sample blanks containing no amplifiedproducts. The standard deviation (SD) of the changes is calculated, anda value obtained by multiplying the SD value by 10 is defined as athreshold value. The number of PCR cycles in which a value larger thanthe threshold value is achieved for the first time referred to as “cyclethreshold value (Ct value)”. Therefore, the larger the initial amount ofa DNA template in a PCR reaction solution, the smaller the Ct value,while the smaller the amount of the template DNA, the larger the Ctvalue. Even if the amounts of the template DNA are the same, the higherthe proportion of the template where a target region of PCR has beencleaved, the larger the Ct value in PCR reactions for the region.

Further, presence or absence of the amplified product can also bedetermined by analyzing the melting temperature (TM) pattern of theamplified product.

All the aforementioned methods can also be used for optimization ofvarious conditions for the method of the present invention.

When live cells of microorganism are detected by the method of thepresent invention, precisions of the determination of the presence orabsence of live cells of microorganism and quantification of the same inthe analysis of the PCR amplified product can be increased by using astandard curve representing relationship between the amount ofmicroorganism and the amplified product, which is prepared by usingstandard samples of the microorganism in which the microorganism isidentified. Although a preliminarily prepared standard curve may beused, it is preferable to use a standard curve prepared by performingthe steps of the method of the present invention for standard samples atthe same time with a test sample. Moreover, if relationship betweenamount of microorganism and amount of DNA or RNA is determinedbeforehand, DNA or RNA isolated from the microorganism can also be usedas a standard sample.

<2> Kit of the Present Invention

The kit of the present invention is a kit for detecting live cells of amicroorganism in a test sample by a nucleic acid amplification method bydistinguishing the live cells from a dead cells or injured cells andcomprises an agent capable of covalently binding to a DNA or RNA byirradiation with light having a wavelength of 350 nm to 700 nm, an agentfor suppressing an action of a nucleic acid amplification inhibitorysubstance, and a primer(s) for amplifying a target region of DNA or RNAof a microorganism to be detected by the nucleic acid amplificationmethod. The kit of the present invention can be used for implementingthe method of the present invention.

In addition, the kit of the present invention may further comprise anyone or more kinds selected from a surfactant, a magnesium salt and anorganic acid salt or a phosphoric acid salt.

Moreover, the kit of the present invention may further comprise anenzyme having an activity of decomposing cells other than that ofmicroorganism, a colloidal particle of a protein, a lipid, or asaccharide existing in the test sample.

The enzyme, the agent capable of covalently binding to a DNA or RNA, andthe agent for suppressing an action of a nucleic acid amplificationinhibitory substance, as well as the surfactant, the magnesium salt andthe organic acid salt or phosphoric acid salt, as required, may be inthe form of a single composition containing all of these components, ortwo or more solutions or compositions containing the components inarbitrary combinations.

The nucleic acid amplification reaction is preferably PCR, LAMP, SDA,LCR, TMA, TRC, HC or microarray method. The cross-linker and the mediumin the kit are the same as those described for the method of the presentinvention.

In a preferred embodiment of the kit of the present invention, the agentcapable of covalently binding to a DNA or RNA is preferably selectedfrom ethidium monoazide, ethidium diazide, propidium monoazide,psoralen, 4,5′,8-trimethylpsoralen, and 8-methoxypsoralen. Ethidiummonoazide is particularly preferably used.

Furthermore, examples of the agent for suppressing an action of anucleic acid amplification inhibitory substance include one or morekinds selected from albumin, dextran, T4 gene 32 protein, acetamide,betaine, dimethyl sulfoxide, formamide, glycerol, polyethylene glycol,soybean trypsin inhibitor, α2-macroglobulin, tetramethylammoniumchloride, lysozyme, phosphorylase, and lactate dehydrogenase.

Furthermore, examples of the magnesium salt include magnesium chloride,magnesium sulfate, magnesium carbonate, and so forth.

Furthermore, examples of the organic acid salt include salts of citricacid, tartaric acid, propionic acid, butyric acid, and so forth.Examples of type of the salt include sodium salt, potassium salt, and soforth. Further, examples of the phosphoric acid salt include salts ofpyrophosphoric acid and so forth. These may be used independently or asa mixture of two or three or more kinds of them.

Furthermore, the enzyme is not particularly limited as long as it candegrade the foreign substances such as cells other than microorganisms,protein colloidal particles, lipids and saccharides existing in the testsample, and does not damage live cells of microorganism to be detected,and examples thereof include lipid-degrading enzymes, protein-degradingenzymes and saccharide-degrading enzymes. Of those, one enzyme or two ormore enzymes may be used, but it is preferable to use both thelipid-degrading enzyme and protein-degrading enzyme, or all of thelipid-degrading enzyme, protein-degrading enzyme andsaccharide-degrading enzyme.

Examples of the lipid-degrading enzymes include lipase, phosphatase, andso forth, examples of the protein-degrading enzymes include serineprotease, cysteine protease, proteinase K, pronase, and so forth, andexamples of the saccharide-degrading enzyme include amylase, cellulase,and so forth.

The kit of the present invention may further comprise a dilutingsolution, a reaction solution for the reaction of the agent capable ofcovalently binding to a DNA or RNA, enzymes and reaction solutions fornucleic acid amplification, an instruction describing the method of thepresent invention, and so forth.

EXAMPLES

Hereinafter, the present invention is described more specifically withreference to the following examples. However, the present invention isnot limited to the examples.

Example 1

By using Enterobacter sakazakii as a representative bacterium ofcoliform bacteria, conditions for definite distinction of live cells anddead cells were examined.

1. Test Materials and Culture Method

1-1) Used Strain and Culture Method

The Enterobacter sakazakii ATCC51329 was cultured at 37° C. for 16 hoursby using the Brain Heart Infusion Broth (BHI broth, Eiken Chemical Co.,Ltd., Tokyo, Japan). The culture broth in a volume of 5 ml was put intoa 15-ml falcon tube (Becton Dickinson Labware, N.J.), and subjected torefrigerated centrifugation at 4° C. and 3,000×G for 10 minutes, thesupernatant was removed, and then 5 ml of physiological saline was addedto the pellet to prepare a stock live cell suspension of Enterobactersakazakii (8.95±0.01 log₁₀ cells/ml, n=2). Further, this live cellsuspension was diluted 10 times with physiological saline to prepare alive cell suspension of Enterobacter sakazakii (7.95±0.01 log₁₀cells/ml, n=2).

Further, 1 ml of the aforementioned stock live cell suspension was putinto a 1.5-ml volume microtube (Eppendorf, Hamburg, Germany), the tubewas immersed into boiling water for 50 seconds and quenched, and it wasconfirmed that the bacterium thereafter did not form any colony on astandard agar medium (Eiken, Tokyo, Japan) to prepare a stock injuredcell suspension of Enterobacter sakazakii. The live cell count ofEnterobacter sakazakii in the live cell suspension was counted on thestandard agar medium, and turbidimetry was simultaneously carried out ata wavelength of 600 nm by using a spectrophotometer U-2800A (Hitachi,Japan) to figure out the relationship between the live cell count andthe turbidity.

Further, the stock live cell suspension was diluted 10 times withmarketed pasteurized cow's milk to prepare a live cell milk suspensionof Enterobacter sakazakii (7.95±0.01 log₁₀ cells/ml, n=2).

Furthermore, the stock injured cell suspension was diluted 10 times withmarketed pasteurized cow's milk to prepare an injured cell milksuspension of Enterobacter sakazakii (7.95±0.01 log₁₀ cells/ml, n=2).

1-2) Ethidium Monoazide (EMA) Treatment and Light Irradiation Treatment

Ethidium monoazide (EMA, Sigma, St. Louis, Mo.) was dissolved at 1000μg/ml using sterilized water, and subjected to filtration sterilizationby using 0.20-μm filter (Minisart-plus, Sartorius AG, Gottingen,Germany) to prepare a stock solution, which was stored at −20° C. underlight shielding.

The EMA solution (1000 μg/ml) in a volume of 10 μl was added to the livecell and injured cell suspensions (1 ml) of Enterobacter sakazakii, andthe suspensions were left at 4° C. for 10 minutes under light shielding.Then, the suspensions were placed at a distance of 20 cm from a visiblelight source (100V PRF 500 W Flood eye, Iwasaki Electric Co., Ltd.,Tokyo, Japan), and irradiated with light for 5 minutes on ice. EachEMA-treated sample was subjected to refrigerated centrifugation at 4° C.and 15,000×G for 10 minutes, the supernatant was removed, then 1 ml ofphysiological saline was added to the pellet for washing, 10 μl ofsterilized water was added to the precipitates (cells) to suspend thecells in the sterilized water, and 5 μl of the suspension was used as asample for PCR amplification.

The live cell and injured cell milk suspensions (1 ml) of Enterobactersakazakii were subjected to an EMA treatment and light irradiationtreatment by the following methods. First, each of the live cell andinjured cell milk suspensions (1 ml) of Enterobacter sakazakii wassubjected to refrigerated centrifugation at 4° C. and 15,000×G for 10minutes, the supernatant was removed, and then 1 ml of physiologicalsaline was added. A protease (derived from Bacillus bacterium, Sigma) ina volume of 3 μl was added to treat the cells with the protease at 37°C. for 1 hour, then the suspension was subjected to refrigeratedcentrifugation (4° C., 15,000×G, 10 minutes), the supernatant wasremoved, 1 ml of physiological saline was added, and then 10 μl of theEMA solution (1000 μg/ml) was added under light shielding. The methodsused thereafter were the same as those described for the aforementioned“live cell and injured cell suspensions of Enterobacter sakazakii (1ml)”.

1-3) PCR Amplification

An agent comprising trisodium citrate dihydrate (TSC, Kanto Kagaku) andmagnesium chloride hexahydrate (Nakarai-Tesque) and further containingone or more selected from bovine serum albumin (BSA, Sigma), dextran(low molecule, M.W.: 50,000 to 70,000, Nakarai-Tesque), T4 gene protein32 (gp32, Nippon Gene), sodium laurylsulfate (SDS, Nakarai-Tesque),Brij56 (Sigma), and egg white lysozyme (Wako Pure Chemical Industries)was added to 5 μl of the sample for PCR amplification. Each agent addedto the sample for PCR amplification and having each composition may alsobe referred to as pretreatment agent. Compositions of the pretreatmentagents are shown below.

Composition 1:

-   2% BSA: 5 μl-   50 mM TSC: 1 μl-   100 mM MgCl₂: 1.5 μl-   0.05% SDS: 1 μl    Composition 2:-   2% BSA: 5 μl-   50 mM TSC: 1 μl-   100 mM MgCl₂: 1.5 μl    Composition 3:-   20% Dextran: 2.5 μl-   50 mM TSC: 1 μl-   100 mM MgCl₂: 1.5 μl    Composition 4:-   0.1% gp32: 5 μl-   50 mM TSC: 1 μl-   100 mM MgCl₂: 1.5 μl    Composition 5:-   2% BSA: 5 μl-   50 mM TSC: 1 μl-   100 mM MgCl₂: 1.5 μl-   4% Brij56: 12.6 μl    Composition 6:-   2% BSA: 5 μl-   50 mM TSC: 1 μl-   100 mM MgCl₂: 1.5 μl-   500 μg/ml Egg white lysozyme: 1.0 μl    Composition 7:-   2% BSA: 5 μl-   50 mM TSC: 1 μl-   100 mM MgCl₂: 1.5 μl-   0.05% SDS: 1 μl-   4% Brij56: 12.6 μl-   500 μg/ml Egg white lysozyme: 1.0 μl    Composition 8:-   2% BSA: 5 μl-   50 mM TSC: 1 μl-   100 mM MgCl₂: 1.5 μl-   4% Brij56: 12.6 μl-   500 μg/ml Egg white lysozyme: 1.0 μl    Composition 9:-   2% BSA: 5 μl    Composition 10:

Only the PCR buffer consisting of the component a) to g) mentionedbelow, not containing the components of the compositions 1 to 9

For the PCR amplification, Primer F: forward primer 16S_10F for 16S rRNAgene detection (5′-AGTTTGATCCTGGCTC-3′, SEQ ID NO: 1), and Primer R:reverse primer 16S_1500R for 16S rRNA gene detection(5′-GGCTACCTTGTTACGA-3′, SEQ ID NO: 2) were used as PCR primers.

Further, in order to maximize variation (primary differential peak) ofthe fluorescent substance amount depending on the temperature to performhigh sensitivity detection in the melt analysis of the amplifiedproducts after the real-time PCR, a PCR buffer consisting of thefollowing components a) to g) was prepared, and the PCR amplificationwas performed by adding this PCR buffer to the mixture of the sample forPCR amplification and the pretreatment agent.

The target of the aforementioned primers was a long DNA (1491 bp)comprising 10 to 1500th nucleotides of 16S rRNA gene.

-   a) Primer F (10 pmol/μl): 4 μl-   b) Primer R (10 pmol/μl): 4 μl-   c) Ex-Taq (5 U/μl, Takara-Bio): 0.5 μl (containing 0.5% Tween 20,    0.5% Nonidet P-40, and 50% glycerol)-   d) 10×Ex-Taq Buffer (Takara-Bio): 5 μl-   e) dNTP mixture (Takara-Bio): 4 μl-   f) 10×SYBR Green I (BMA): 8 μl-   g) Sterilized water: volume required to obtain the total volume of    55 μl including 5 μl of the sample for PCR amplification and the    pretreatment agent

Real-time PCR was performed according to the following PCR thermal cycleconditions by using a real-time PCR apparatus (iCycler iQ, Bio-Rad,Hercules, Calif.).

-   1) 4° C. for 3 minutes (1 cycle)-   2) 94° C. for 30 seconds (1 cycle)-   3) 94° C. for 20 seconds; 55° C. for 30 seconds; 72° C. for 1 minute    and 30 seconds (50 cycles)-   4) 95° C. for 3 minutes (1 cycle)

Then, according to the protocol of the melt analysis of the PCRamplified product (temperature was raised at intervals of 0.1° C. from60° C., each temperature was maintained for 8 seconds, and thisprocedure was repeated 350 times in total up to the final temperature of95° C.), the melting temperature of the PCR amplified product wasmeasured.

As a positive control, 8 log₁₀ cells/ml live cell suspension ofEnterobacter sakazakii was used. Further, as a blank sample, the PCRbuffer itself to which nothing was added was used for PCR.

2. Results

The results of the real-time PCR are shown in Table 1.

The symbols a) to f) used in Table 1 indicate the followings. Further,“Lyso” used for the pretreatment agent means egg white lysozyme.

-   a) Live cell counts and injured cell counts of Enterobacter    sakazakii: 7.95±0.01 log₁₀ cells/ml (in physiological saline and    marketed pasteurized cow's milk)-   b) Injured cells were prepared by immersing live cells in boiling    water for 50 seconds.-   c) No treatment with EMA is meant.-   d) EMA treatment (10 μg/ml, 10 minutes, 4° C. under light    shielding)+visible light irradiation (5 minutes)-   e) Ct value of real-time PCR indicated as mean±SD (n=2)-   f) nd means that amplification of the objective gene was not    attained by the real-time PCR.

TABLE 1 In physiological saline Pretreatment agent Live cell^(a))Composition BSA Dex Gp32 TSC MgCl₂ SDS Brij56 Lyso 0^(c)) 10^(d)) 1 + −− + + + − − 17 ± 0.9 21 ± 3.2 2 + − − + + − − − 17 ± 0.6 20 ± 1.4 3 − +− + + − − − 17 ± 0.7 20 ± 0.8 4 − − + + + − − − 18 ± 0.5 20 ± 1.8 5 + −− + + − + − 16 ± 0.8 19 ± 0.9 6 + − − + + − − + 17 ± 1.1 19 ± 0.7 7 + −− + + + + + 15 ± 0.6 18 ± 1.2 8 + − − + + − + + 16 ± 0.4 19 ± 1.3 9 + −− − − − − − 18 ± 0.8 21 ± 1.1 10  − − − − − − − − 25 ± 0.7 28 ± 1.1 Inphysiological saline In cow's milk Injured cell^(a)b)) Live cell^(a))Injured cell^(a)b)) Composition 0 10 0^(c)) 10^(d)) 0 10 1 20 ± 4.2^(e))nd^(f)) 32 ± 0.8 35 ± 0.9 35 ± 1.1 nd 2 20 ± 1.1 nd 31 ± 1.4 34 ± 1.6 35± 1.0 nd 3 20 ± 1.2 nd 32 ± 0.7 34 ± 1.4 35 ± 1.2 nd 4 20 ± 1.8 nd 33 ±0.6 35 ± 0.6 35 ± 1.8 nd 5 19 ± 0.8 nd 25 ± 1.2 27 ± 0.8 27 ± 1.4 nd 619 ± 0.6 nd 28 ± 0.9 31 ± 0.8 32 ± 0.9 nd 7 18 ± 0.8 nd 22 ± 0.8 24 ±0.5 24 ± 1.5 nd 8 19 ± 1.2 nd 22 ± 0.8 24 ± 0.8 24 ± 1.2 nd 9 21 ± 1.3nd 34 ± 1.1 37 ± 1.4 38 ± 1.6 nd 10  28 ± 1.8 nd nd nd nd nd

As seen from the results for the compositions 1 and 2 shown in Table 1,in this system where PCR was directly performed in the bacterium,distinction of live state and injured state (distinction of live cellsand injured cells) of Enterobacter sakazakii in physiological saline,and distinction of live state and injured state of Enterobactersakazakii in cow's milk were clearly attained, and according to theevaluation based on the Ct value serving as an index of the reactionrate of the real-time PCR, significant differences were not observedamong the Ct values of Enterobacter sakazakii in physiological salineand cow's milk (live cells, not treated with EMA), or among those of thelive cells subjected to the EMA treatments of various conditions. Fromthese results, it was found that it makes no difference if thesurfactant SDS is contained or not contained in the pretreatment agent.The same phenomenon as mentioned above was also observed in comparisonof the results obtained with the compositions 7 and 8.

As seen from comparison of the results obtained with the compositions 2,3 and 4, for all the cases where any of albumin, dextran, and the T4gene protein 32 was contained in the composition, distinction of livestate and injured state of Enterobacter sakazakii (in physiologicalsaline and cow's milk) was clearly attained, and significant differenceswere not observed among the Ct values of Enterobacter sakazakii inphysiological saline and cow's milk (live cells, not treated with EMA),or among those of the live cells subjected to the EMA treatments ofvarious conditions. On the basis of these data, it was found that any ofalbumin, dextran, and the T4 gene protein 32 may be used.

As seen from comparison of the results obtained with the compositions 2and 5, distinction of live state and injured state of Enterobactersakazakii was clearly attained with both compositions (in physiologicalsaline and cow's milk), but it was suggested that addition of thenonionic surfactant Brij56 provided a significant decrease of the Ctvalues of Enterobacter sakazakii (live cells, not treated with EMA andtreated with EMA), especially that of the cells in cow's milk, and thussensitivity for detection of live cells was improved. Furthermore, incomparison of the results obtained with the compositions 2 and 6, it wasobserved that distinction of live state and injured state was clearlyattained with both compositions (in physiological saline and cow'smilk), but there was a tendency that addition of lysozyme more improvedthe sensitivity for detection of live cells in cow's milk (not treatedwith EMA and treated with EMA).

On the basis of comparison of the results obtained with the compositions5, 6 and 8, it was found that distinction of live state and injuredstate was clearly attained with all of the compositions (inphysiological saline and cow's milk), but coexistence of Brij56 and eggwhite lysozyme clearly improved the sensitivity for detection of livecells (not treated with EMA and treated with EMA) as shown by theresults obtained with the composition 8. Egg white lysozyme directlyacts on the peptidoglycans of gram-positive bacteria to hydrolyze thepolysaccharides (β-1,4-linkages of N-acetylglucosamine andN-acetylmuramic acid), however, in the case of gram-negative bacteria,there is an outer membrane outside the peptidoglycan containing thesepolysaccharides (on the side where egg white lysozyme functions), andtherefore egg white lysozyme cannot act on. Taking this action mechanisminto consideration, it can be considered that egg white lysozyme in thecomposition 8 did not promote lysis (disruption) of the cells ofEnterobacter sakazakii, which is a gram-negative bacterium, but stronglyacted on cell walls of dead cells of gram-positive bacteria existing incow's milk in advance (≧5 log₁₀ cells/ml) in the presence of Brij56 tophysicochemically change the cell wall surface structures ofgram-positive bacteria, which are conventionally considered to be PCRinhibitory components, and therefore the cell wall surface structurescould no longer function as PCR inhibitory components. In fact, incomparison of the results obtained with the compositions 5, 6 and 8, itcan be seen that the sensitivity for detection of live cells in cow'smilk was significantly improved with the composition 8, butgram-positive bacteria did not exist as contaminants among the livecells suspended in physiological saline, and any data indicating thatthe composition 8 especially promotes reactions in PCR have not beenobtained for live cells of Enterobacter sakazakii, which is agram-negative bacterium.

From the above, it can be considered that egg white lysozyme did notparticipate in the lysis of gram-negative bacteria in the presence ofBrij56, but physicochemically changed the cell walls of thegram-positive bacteria originally contained in the test sample andconsidered as PCR contaminants to make them not function as PCRinhibitory components, and therefore it markedly improved thesensitivity for detection of live cells of Enterobacter sakazakii as theobject, which is a gram-negative bacterium, as a result. This alsoagrees with the fact that, in comparison of the results obtained withthe compositions 2 and 8, any significant difference was not observed inthe sensitivity (Ct value) for detecting live cells in physiologicalsaline, but extremely superior sensitivity for detecting live cells incow's milk (containing many injured cells and dead cells ofgram-positive bacteria) was obtained with the composition 8. Moreover,it is considered that egg white lysozyme adsorbs to a nucleic acidamplification inhibitory substance to normally advance the nucleic acidamplification reaction.

On the basis of comparison of the results obtained with the compositions1, 2 and 9, it is considered that if the function of a nucleic acidamplification reaction inhibitory substance can be suppressed with BSA,distinction of live cells and injured cells is possible, even if amagnesium salt or an organic acid salt is not contained. In addition,with the composition 9, the Ct values for the live cells (not treatedwith EMA and treated with EMA) and injured cells (not treated with EMA)delayed by about 3, and therefore the compositions 1 and 2 containingthe magnesium salt or the organic acid salt are more advantageous inview of the reactivity.

Finally, as seen from comparison of the results obtained with thecompositions 2 and 10, although distinction of live state and injuredstate of Enterobacter sakazakii suspended in physiological saline waspossible only with the PCR buffer, but the sensitivity (Ct value) forlive cells (not treated with EMA and treated with EMA) was extremelysignificantly unfavorable, in addition, in the case of supposing cow'smilk as a usual typical test sample, live cells (not treated with EMAand treated with EMA) and injured cells not treated with EMA cannot bedetected only with the PCR buffer, and therefore it is considered thatit is preferred that at least an agent that can reduce the influence ofa PCR inhibitory substance, of which representative example is albumin,and a magnesium salt and an organic acid salt or a phosphoric acid saltare contained.

Example 2

Distinctions of live cells and injured cells of coliform bacteria andEnterobacteriaceae bacteria were performed.

1. Test Materials and Test Methods

1-1) Used Strains and Culture Method

Bacteria of 16 genera of the coliform bacteria and 1 genus ofEnterobacteriaceae not belonging to the coliform bacteria, Klebsiellaoxytoca JCM1665, Citrobacter koseri JCM1658, Enterobacter sakazakiiATCC51329, Serratia fonticola JCM1242, Budvicia aquilia JCM3902,Rahnella aquatilis NBRC13544, Hafnia alvei JCM1666, Leclerciaadecarboxylata JCM1667, Yokenella regensburgei JCM2403, Pantoeaagglomerans JCM1236, Buttiauxella agrestis JCM1090, Kluyvera ascorbataJCM2107, Cedecea davisae JCM1685, E. coli (Escherichia coli) DH5α, andSalmonella enteritidis IID604 were cultured at 37° C. for 16 hours byusing the Brain Heart Infusion Broth (BHI broth, Eiken Chemical Co.,Ltd., Tokyo, Japan).

Further, bacteria of 2 species belonging to 2 genera of the coliformbacteria, Ewingella americana JCM4911 and Moellerella wisconsensisJCM5894 were cultured at 30° C. for 16 hours by using the BHI broth.

Each culture broth in a volume of 5 ml after the culture was collectedin a 15-ml falcon tube (Becton Dickinson Labware, N.J.), and subjectedto refrigerated centrifugation at 4° C. and 3,000×G for 10 minutes, thesupernatant was removed, then 5 ml of physiological saline was added tothe precipitates (pellet), and the suspension was diluted 10 times withphysiological saline to prepare a live cell suspension of each strain.

Further, 1 ml of the aforementioned live cell suspension was put into a1.5-ml volume microtube (Eppendorf, Hamburg, Germany), and the tube wasimmersed into boiling water for 50 seconds and quenched. It wasconfirmed that each suspension immersed in boiling water did not formany colony on the standard agar medium (Eiken, Tokyo, Japan) to prepareinjured cell suspensions of the coliform bacteria and Enterobacteriaceaebacteria.

The live cell suspensions and injured cell suspensions prepared asdescribed above were used as test samples for the following tests.

The live cell counts of the coliform bacteria and Enterobacteriaceaebacteria in the live cell suspensions were counted on the standard agarmedium, and turbidimetry was simultaneously carried out at a wavelengthof 600 nm by using a spectrophotometer U-2800A (Hitachi, Japan) tofigure out the relationship between the live cell count and theturbidity.

1-2) Ethidium Monoazide (EMA) Treatment and Light Irradiation Treatment

Ethidium monoazide (EMA, Sigma, St. Louis, Mo.) was dissolved at 1000μg/ml using sterilized water, and subjected to filtration sterilizationby using 0.20-μm filter (Minisart-plus, Sartorius AG, Gottingen,Germany) to prepare a stock solution (EMA solution), which was stored at−20° C. under light shielding.

The EMA solution (1000 μg/ml) in a volume of 10 μl was added to eachtest sample (live cell suspension or injured cell suspension) in avolume of 1 ml, and the mixture was left at 4° C. for 10 minutes underlight shielding.

Then, the test sample was set at a distance of 20 cm from a visiblelight source (100V PRF 500 W Flood eye, Iwasaki Electric Co., Ltd.,Tokyo, Japan), and irradiated with visible light for 5 minutes on ice.

The EMA-treated and visible light-irradiated test sample was subjectedto refrigerated centrifugation at 4° C. and 15,000×G for 10 minutes, thesupernatant was removed, then 1 ml of physiological saline was added tothe pellet for washing, and the suspension was further subjected torefrigerated centrifugation to collect the precipitates. Such a washingtreatment was repeated several times, and then 10 μl of sterilized waterwas added to the precipitates (cells) to suspend the cells in thesterilized water to prepare a sample for PCR amplification.

1-3) PCR Amplification

An agent comprising bovine serum albumin (BSA, Sigma), trisodium citratedihydrate (TSC, Kanto Kagaku), and magnesium chloride hexahydrate(Nakarai-Tesque) in the compositions 1) to 3) mentioned below was addedto each sample for PCR amplification, and a surfactant of 4) comprisingsodium laurylsulfate (SDS, Nakarai-Tesque) was added to 5 μl of thesample for PCR amplification.

In the following descriptions, a combination consisting of the agenthaving the compositions of 1) to 3) and the surfactant of 4) may also bereferred to as a pretreatment agent.

-   1) 2% BSA: 5 μl-   2) 50 mM TSC: 1 μl-   3) 100 mM MgCl₂: 1.5 μl-   4) 0.05% SDS: 1 μl

For the PCR amplification, Primer F: forward primer 16S_10F for 16S rRNAgene detection (SEQ ID NO: 1), and Primer R: reverse primer 16S_1500Rfor 16S rRNA gene detection (SEQ ID NO: 2) were used as PCR primers.

Further, in order to maximize variation (primary differential peak) ofthe fluorescent substance amount depending on the temperature to performhigh sensitivity detection in the melt analysis of the amplifiedproducts after the real-time PCR, a PCR buffer consisting of thefollowing components a) to g) was prepared, and the PCR amplificationwas performed by adding this PCR buffer to the mixture of the sample forPCR amplification and the pretreatment agent.

-   a) Primer F (10 pmol/μl): 4 μl-   b) Primer R (10 pmol/μl): 4 μl-   c) Ex-Taq (5 U/μl, Takara-Bio): 0.5 μl (containing 0.005% Tween 20    and 0.005% Nonidet P-40)-   d) 10×Ex-Taq Buffer (Takara-Bio): 5 μl-   e) dNTP mixture (Takara-Bio): 4 μl-   f) 10×SYBR Green I (BMA): 8 μl-   g) Sterilized water: 16 μl

Real-time PCR was performed according to the following PCR thermal cycleconditions by using a real-time PCR apparatus (iCycler iQ, Bio-Rad,Hercules, Calif.).

-   1) 4° C. for 3 minutes (1 cycle)-   2) 94° C. for 30 seconds (1 cycle)-   3) 94° C. for 20 seconds; 55° C. for 30 seconds; 72° C. for 90    seconds (50 cycles)-   4) 95° C. for 3 minutes (1 cycle)

Then, according to the protocol of the melt analysis of the PCRamplified product (temperature was raised at intervals of 0.1° C. from60° C., each temperature was maintained for 8 seconds, and thisprocedure was repeated 350 times in total up to the final temperature of95° C.), the melting temperature of the PCR amplified product wasmeasured.

As a positive control, live cell suspension of Enterobacter sakazakii (8log₁₀ cells/ml) was used to perform PCR amplification in the samemanner. Further, as a blank sample, the PCR buffer itself to which anytest sample was not added was used to perform PCR amplification.

1-4) Gel Electrophoresis

2% Agarose gel (2% Seakem GTG agarose, FCM BioProducts, Rockland, Me.)was prepared by using 0.5×TAE.

The PCR amplified product was applied in a volume of 10 μl to theagarose gel, and electrophoresis was performed.

The gel was stained with 1 μg/ml ethidium bromide solution, and observedas a densitograph, and the image thereof was captured and stored byusing AE-6905H Image Saver HR (Atto Co., Japan).

2. Results

The results of the real-time PCR are shown in Table 2. Further, theresults of the electrophoresis of the final PCR amplified products areshown in FIG. 1.

The meanings of the symbols used in FIG. 1 are as follows.

-   EMA+: EMA treatment (10 μg/ml, 10 minutes, 4° C. under light    shielding)+visible light irradiation (5 minutes)-   EMA−: No EMA treatment-   PC: Positive control in which 8 log₁₀ cells/ml live cell suspension    of Enterobacter sakazakii was used in a volume of 5 μl-   NC: Negative control in which sterilized water was used instead of    the DNA template-   M: 100 bp DNA ladder-   Injured cell: Injured cells prepared by immersing the live cell    suspension in boiling water for 50 seconds was used.

The cell counts in the live cell suspensions of each strain is asfollows.

-   E. coli: Escherichia coli DH5α (7.91±0.20 log₁₀ cells/ml)-   S. enteritidis: Salmonella enteritidis IIP 604 (8.07±0.02 log₁₀    cells/ml)-   K. oxytoca: Klebsiella oxytoca JCM1665 (8.38±0.08 log₁₀ cells/ml)-   C. koseri: Citrobacter koseri JCM1658 (8.02±0.06 log₁₀ cells/ml)-   E. sakazakii: Enterobacter sakazakii ATCC51329 (7.95±0.01 log₁₀    cells/ml)-   S. fonticola: Serratia fonticola JCM1242 (7.47±0.01 log₁₀ cells/ml)-   B. aquilia: Budvicia aquilia JCM3902 (6.98±1.50 log₁₀ cells/ml)-   R. aquatilis: Rahnella aquatilis NBRC13544 (7.38±0.14 log₁₀    cells/ml)-   E. americana: Ewingella americana JCM4911 (7.47±0.43 log₁₀ cells/ml)-   H. alvei: Hafnia alvei JCM1666 (8.04±0.22 log₁₀ cells/ml)-   L. adecarboxylata: Leclercia adecarboxylata JCM1667 (7.46±0.20 log₁₀    cells/ml)-   M. wisconsensis: Moellerella wisconsensis JCM5894 (7.85±0.34 log₁₀    cells/ml)-   Y. regensburgei: Yokenella regensburgei JCM2403 (8.03±0.13 log₁₀    cells/ml)-   P. agglomerans: Pantoea agglomerans JCM1236 (7.67±0.78 log₁₀    cells/ml)-   B. agrestis: Buttiauxella agrestis JCM1090 (7.76±0.00 log₁₀    cells/ml)-   K. ascorbata: Kluyvera ascorbata JCM2107 (7.80±0.02 log₁₀ cells/ml)-   C. davisae: Cedecea davisae JCM1685 (7.56±0.10 log₁₀ cells/ml).

TABLE 2 Coliform bacteria/Enterobacteriaceae Live cell ^(a)) Injuredcell ^(b)) bacteria log₁₀ cells/ml 0 ^(c)) 10 ^(d)) 0 10 Klebsiellaoxytoca JCM1665 8.38 ± 0.08 19 ± 0.5 20 ± 1.3 20 ± 1.2 n.d. ^(f))Citrobacter koseri JCM1658 8.02 ± 0.06 18 ± 0.8 19 ± 0.7 19 ± 1.3 n.d.Escherichia coli DH5 α 7.91 ± 0.20 22 ± 0.8 ^(e)) 23 ± 0.5 20 ± 1.3 n.d.Salmonella enteritidis IIP 604 8.47 ± 0.02 19 ± 1.4 19 ± 0.6 18 ± 0.8n.d. Enterobacter sakazakii ATCC 51329 7.95 ± 0.01 17 ± 0.9 21 ± 3.2 20± 4.2 n.d. Serratia fonticola JCM 1242 7.47 ± 0.01 17 ± 1.6 20 ± 1.2 18± 1.1 n.d. Budvicia aquilia JCM 3902 6.98 ± 1.50 16 ± 3.2 24 ± 1.7 15 ±2.0 n.d. Rahnella aquatilis NBRC 13544 7.38 ± 0.14 20 ± 0.4 23 ± 0.0 21± 1.4 n.d. Ewingella americana JCM 4911 7.47 ± 0.43 15 ± 0.2 17 ± 0.4 17± 0.1 n.d. Hafnia alvei JCM1666 8.04 ± 0.22 17 ± 0.2 18 ± 1.6 16 ± 1.3n.d. Leclercia adecarboxylata E1667 7.46 ± 0.20 13 ± 0.5 16 ± 1.0 16 ±1.5 n.d. Moellerella wisconsensis JCM 5894 7.85 ± 0.34 15 ± 2.7 19 ± 5.616 ± 1.3 n.d. Yokenella regensburgei JCM 2403 8.03 ± 0.13 17 ± 0.2 16 ±0.4 15 ± 0.4 n.d. Pantoea agglomerans JCM 1236 7.67 ± 0.78 15 ± 0.1 16 ±1.5 16 ± 0.2 n.d. Buttiauxella agrestis JCM 1090 7.76 ± 0.00 13 ± 1.0 17± 1.1 18 ± 2.8 n.d. Kluyvera ascorbata JCM 2107 7.80 ± 0.02 17 ± 1.4 18± 0.8 18 ± 0.4 n.d. Cedecea davisae JCM 1685 7.56 ± 0.10 21 ± 1.4 24 ±2.1 22 ± 1.4 n.d. The symbols ^(a)) to ^(f)) used in Table 2 have thefollowing meanings. ^(a)) Live cell counts of the coliformbacteria/Enterobacteriaceae bacteria numerical values in the columnsmean Ct values in real-time PCR. ^(b)) Injured cells were prepared byimmersing the live cell suspension in boiling water for 50 seconds.^(c)) No treatment with EMA is meant. ^(d)) EMA final concentration of10 μg/ml is meant. ^(e)) Ct value is indicated as mean ± SD (n = 2).^(f)) n.d. means that PCR was performed twice, but the objective genedid not amplified in both PCRs.

As shown in the results of Table 2, the Ct values (number of cyclescorresponding to rising of real-time PCR curve) of the EMA-untreatedgroup of live cells were 13 to 22, and the Ct values of the EMA-treatedgroup of live cells were 16 to 24. Further, the Ct values of theEMA-untreated group of injured cells were 15 to 22, and all the groupsprovided good PCR amplification results. However, in the EMA-treatedgroups of injured cells of all the coliform bacteria andEnterobacteriaceae bacteria, amplification of the target gene was notattained.

Furthermore, as shown in the results of the electrophoresis shown inFIG. 1, any band indicating positive result for the PCR amplifiedproduct could not be detected only for the EMA-treated groups of injuredcells of all the coliform bacteria and Enterobacteriaceae bacteria.

On the basis of the above results, it became clear that, by carrying outthe method of the present invention, distinction of live cells andinjured cells as well as distinction of live cells and dead cells areenabled for 16 genera of coliform bacteria and 2 genera of the familyEnterobacteriaceae. Further, distinction of live cells and injured cellswas also made possible for species of which detection has conventionallybeen difficult, for example, Escherichia bacteria and Salmonellabacteria.

Example 3

Distinctions of live cells and injured cells of coliform bacteria andEnterobacteriaceae bacteria inoculated into a foodstuff, i.e., cow'smilk, were performed.

1. Test Materials and Test Methods

1-1) Used Strains and Culture Method

Bacteria of 1 genus of the family Enterobacteriaceae and 12 genera ofthe coliform bacteria, Kluyvera ascorbata JCM2107, Cedecea davisaeJCM1685, Citrobacter koseri JCM1658, Klebsiella pneumoniae NRBC3321,Serratia fonticola JCM1242, Yokenella regensburgei JCM2403, Rahnellaaquatilis NBRC13544, Hafnia alvei JCM1666, Leclercia adecarboxylataJCM1667, Pantoea agglomerans JCM1236, Enterobacter sakazakii ATCC51329,E. coli DH5α, and Salmonella enteritidis IID604 were cultured at 37° C.for 16 hours by using the Brain Heart Infusion Broth (BHI broth, EikenChemical Co., Ltd., Tokyo, Japan).

Each culture broth in a volume of 5 ml after the culture was collectedin a 15-ml falcon tube (Becton Dickinson Labware, N.J.), and subjectedto refrigerated centrifugation at 4° C. and 3,000×G for 10 minutes, thesupernatant was removed, then 5 ml of physiological saline was added tothe precipitates (pellet), and the suspension was diluted 10 times withphysiological saline to prepare a live cell suspension of each strain.

The live cell suspensions prepared as described above were used as testsample for the following tests.

The live cell counts of the coliform bacteria and Enterobacteriaceaebacteria in the live cell suspensions were counted on the standard agarmedium, and turbidimetry was simultaneously carried out at a wavelengthof 600 nm by using a spectrophotometer U-2800A (Hitachi, Japan) tofigure out the relationship between the live cell count and theturbidity.

1-2) Inoculation of Bacterium Suspension to Foodstuff and Collection ofPrecipitates

To 22.2 ml of marketed pasteurized cow's milk (live cells were notdetected by the cultivation method), 9 to 25 cells were inoculated byusing the live cell suspensions of various coliform bacteria andEnterobacteriaceae bacteria prepared above.

To 22.2 ml of the cow's milk, one kind of coliform bacterium orEnterobacteriaceae bacterium was inoculated.

Further, as a sample blank, 22.2 ml of cow's milk to which any live cellsuspension was not added was prepared (bacterium not inoculated).

The cow's milk to which live cells of coliform bacteriumEnterobacteriaceae bacterium was inoculated and the cow's milk to whichany bacterium was not inoculated, as prepared above, were, subjected tocentrifugation at 37° C. and 3,000×G for 5 minutes, and the lipid layeron the surface of the supernatant and the aqueous layer existing as aninterlayer were removed by decantation to collect the precipitates.

The collected precipitates (pellets) of the cow's milk to which livecells of coliform bacterium or Enterobacteriaceae bacterium wasinoculated, and to which any bacterium was not inoculated both,contained dead cells of bacteria originally existed in the marketedcow's milk and made extinct by sterilization (gram-negative bacteria orgram-positive bacteria including coliform bacteria etc. (≧6 log₁₀cells)).

Therefore, it was judged that the precipitates prepared from the cow'smilk to which live cells of the coliform bacterium or Enterobacteriaceaebacterium was inoculated contained dead cells and live cells.

1-3) Enzyme Treatment

To each of the precipitates (test sample) prepared from the cow's milkto which live cells of the coliform bacterium or Enterobacteriaceaebacterium as prepared above, 10 ml of the Brain Heart Infusion (BHI)broth kept warm at 37° C. beforehand was added, and the cells weresuspended therein. To the suspension, 25 μl of a diluted enzyme solutionprepared by diluting a proteinase K solution (equivalent to 1250 U/ml,EC.3.4.21.64, Sigma) 50 times with physiological saline (25 U/ml) wasadded to perform an enzyme treatment at 37° C. for 3 hours.

The enzyme-treated test sample was centrifuged at 37° C. and 3,000×G for5 minutes, the supernatant was removed, and the precipitates werecollected again.

1-4) Ethidium Monoazide (EMA) Treatment and Light Irradiation Treatment

After 1 ml of physiological saline was added to the precipitates afterthe enzyme treatment, and the mixture was stirred, 10 μl of an EMAsolution (1000 μg/ml) prepared in the same manner as that of Example 2was added to the mixture, and the mixture was left at 4° C. for 10minutes under light shielding.

Then, visible light irradiation and washing treatment were performed inthe same manner as that of Example 2, and 5 μl of sterilized water wasadded to the precipitates to prepare a sample for PCR amplification.

1-5) PCR Amplification

A pretreatment agent was added to 5 μl of the sample for PCRamplification as in Example 2.

For the PCR amplification, Primer F: forward primer 16S_1234F for 16SrRNA gene detection (5′-CTACAATGGCGCATACAAAGAGAAG-3′, SEQ ID NO: 3), andPrimer R: reverse primer 23S_1703R for 23S rRNA gene detection(5′-CCTTCTCCCGAAGTTACGGCACCAT-3′, SEQ ID NO: 4) were used as PCRprimers.

Further, in order to maximize variation (primary differential peak) ofthe fluorescent substance amount depending on the temperature to performhigh sensitivity detection in the melt analysis of the amplifiedproducts after the real-time PCR, a PCR buffer comprising the followingcomponents a) to g) was prepared, and PCR amplification was performed byadding 41.5 μl of this PCR buffer to the mixture of the sample for PCRamplification and the pretreatment agent.

The PCR primers contained the nucleotides of the 1234 to 1258 positionsof the 16S rRNA gene, a tRNA gene (76 bp) and the nucleotides of thepositions 1 to 1703 of the 23S rRNA gene, and the target thereof was along DNA (about 2450 bp) containing a spacer region (about 364 bp).

-   a) Primer F (10 pmol/μl): 4 μl-   b) Primer R (10 pmol/μl): 4 μl-   c) Ex-Taq (5 U/μl, Takara-Bio): 0.5 μl-   (containing 0.5% Tween 20, 0.5% Nonidet P-40, and 50% glycerol)-   d) 10×Ex-Taq Buffer (Takara-Bio): 5 μl-   e) dNTP mixture (Takara-Bio): 4 μl-   f) 10×SYBR Green I (BMA): 8 μl-   g) Sterilized water: 16 μl

Real-time PCR was performed according to the following PCR thermal cycleconditions by using a real-time PCR apparatus (iCycler iQ, Bio-Rad,Hercules, Calif.).

-   1) 95° C. for 3 minutes (1 cycle)-   2) 95° C. for 30 seconds; 60° C. for 40 seconds; 68° C. for 3    minutes (40 cycles)-   3) 95° C. for 3 minutes (1 cycle)

Then, according to the protocol of the melt analysis of the PCRamplified product (temperature was raised at intervals of 0.1° C. from60° C., each temperature was maintained for 8 seconds, and thisprocedure was repeated 350 times in total up to the final temperature of95° C.), the melting temperature of the PCR amplified product wasmeasured.

As a positive control, 8 log₁₀ cells/ml live cell suspension ofEnterobacter sakazakii was used to perform PCR amplification in the samemanner. Further, as a blank sample, the PCR buffer itself to which anytest sample was not added was used to perform PCR amplification.

1-6) Gel Electrophoresis

0.8% Agarose gel (Seakem GTG agarose, FCM BioProducts, Rockland, Me.)was prepared by using 0.5×TAE.

The PCR amplified product in a volume of 5 to 10 μl was applied to theagarose gel to perform electrophoresis.

In a solution prepared by diluting SYBR Gold nucleic acid gel stain(Invitrogen, Eugene, Oreg., USA) 10,000 times with 0.5×TAE, the agarosegel after the electrophoresis was immersed for 15 minutes and therebystained, then the gel was observed as a densitograph, and the imagethereof was captured and stored by using AE-6905H Image Saver HR (AttoCo., Japan).

2. Results

The results of the real-time PCR are shown in Table 3. Further, theresults of the electrophoresis of the final PCR amplified products areshown in FIG. 2.

The meanings of the symbols used in FIG. 2 are as follows.

-   KP: K. pneumoniae-   CK: C. koseri-   EC: E. coli.-   SE: S. enteritidis-   KA: K. ascorbata-   CD: C. davisae-   SF: S. fonticola-   YR: Y. regensburgei-   RA: R. aquatilis-   HA: H. alvei-   LA: L. adecarboxylata-   PA: P. agglomerans-   ES: E. sakazakii-   Milk: Marketed pasteurized cow's milk not inoculated with coliform    bacterium-   Positive: Positive control (Enterobacter sakazakii, 5 μl of 8 log₁₀    CFU/ml was used as a template for PCR)-   Negative: Negative control (5 μl of sterilized water was used as a    PCR template)-   L: 100 bp DNA ladder.

TABLE 3 Real time PCR Coliform Live cell count^(a)) [logCFU/sample]Electrophoresis/ bacteria/Enterobacteriaceae After incubation Δ cell TMpattern^(b)) gel staining^(c)) bacteria Initial for 3 hours count F = 5F = 10 F = 5 F = 10 Klebsiella pneumoniae NBRC 3321 1.4 ± 0.0 3.4 ± 0.22.0 4/4 4/4 4/4 4/4 Citrobacter kosei JCM 1658 1.4 ± 0.0 3.3 ± 0.1 1.92/2 1/2 2/2 1/2 Kluyvera ascorbata JCM 21070 1.2 ± 0.0 4.4 ± 1.1 3.2 2/22/2 2/2 2/2 Escherichia coli DH5α 1.1 ± 0.1 2.3 ± 0.1 1.2 2/2 1/2 2/22/2 Cedecea davisae JCM 1685 1.0 ± 0.1 2.7 ± 0.4 1.7 0/2 0/2 2/2 2/2Salmonella enteritidis IID 604 1.4 ± 0.1 3.5 ± 0.2 2.1 2/2 1/2 2/2 2/2Serratia fonticola JCM 1242 0.9 ± 0.1 2.3 ± 0.1 1.4 1/2 1/2 2/2 1/2Yokenella regensburgei JCM 2403 1.0 ± 0.5 3.0 ± 0.4 2.0 2/2 2/2 2/2 2/2Rahnella aquatilis NBRC 13544 1.0 ± 0.0 2.3 ± 0.4 1.3 1/2 2/2 2/2 2/2Hafnia alvei JCM 1666 1.3 ± 0.1 3.3 ± 0.6 2.0 2/2 2/2 2/2 2/2 Leclerciaadecarboxylata JCM 1667 0.8 ± 0.0 2.6 ± 0.3 1.8 2/2 2/2 2/2 2/2 Pantoeaagglomerans JCM 1236 1.1 ± 0.3 2.2 ± 0.4 1.1 0/2 2/2 2/2 2/2Enterobacter sakazakii ATCC 51329 1.2 ± 0.2 3.1 ± 0.2 1.8 8/8 8/8 8/88/8 Average or tatal 1.1 ± 0.2 2.9 ± 0.6 1.8 ± 0.6 28/34 28/34 34/3432/34 Meanings of the symbols ^(a)) to ^(c)) used in Table 3 are asfollows. ^(a))Live cell counts of the coliform bacteria, the measurementwas performed with n = 2 to 8. ^(b))Number of positive results to thetotal number of the measurement of the amplified product by the meltanalysis (TM pattern) based on real-time PCR ^(c))Number of positiveresults to the total number of the measurement of the PCR amplifiedproduct by the electrophoresis and gel staining (SYBR Gold) method.

From the results shown in Table 3, in the samples prepared byinoculating 9 to 25 cells of the various coliform bacteria (live cells)in 22.2 ml of pasteurized cow's milk, then centrifuging the milk, andperforming a proteinase K treatment in the BHI broth and incubation(proliferation step of live coliform bacteria), the number of thecoliform bacteria (live cells) increased to 90 to 2,900 cells.

Further, as a result of performing melt analysis of the PCR amplifiedproducts and electrophoresis of the PCR amplified products, it waspossible to detect live cells for all the 13 strains. In addition, thePCR amplification product obtained from the cow's milk not inoculatedwith coliform bacteria (live cells) showed a negative result in both themelt analysis and the electrophoresis.

From the above results, it was revealed that it was made possible todetect live cells of coliform bacteria and Enterobacteriaceae bacteriainoculated into a foodstuff such as cow's milk by distinguishing themfrom dead cells (detection of live cells) by the method of the presentinvention. Therefore, it became possible to widely apply suchdistinction to foodstuffs such as cow's milk and detect live cells withhigh sensitivity, even for various cells for which distinction of livecells and dead cells have been difficult (bacteria, viruses, etc.).

Control Example 1 Detection of Live Cells by Conventional Method

High concentration of injured cells of coliform bacterium (alsoincluding Enterobacteriaceae bacteria cells) were detected by theEMA-PCR method targeting 16S rRNA (long DNA) in which cells weresubjected to an EMA treatment, and then DNA was purified by DNAextraction and used as a template.

1. Test Materials and Test Methods

1-1) Used Strains and Culture Method

The method of this test was carried out according to the method ofJapanese Patent No. 4217797 (International Patent Publication No.WO2002/052034).

Escherichia coli DH5α, Salmonella enteritidis IID604, Klebsiella oxytocaJCM1665, and Citrobacter koseri JCM1658 were cultured at 37° C. by usingthe Brain Heart Infusion Broth (BHI broth, Eiken, Tokyo).

A predetermined volume (10 ml) aliquot was collected from each culturebroth in which the cells were in the logarithmic phase, and subjected torefrigerated centrifugation at 4° C. and 8,000×G for 15 minutes. Afterthe supernatant was removed, 10 ml of physiological saline was added tothe pellet to suspend the cells again, a similar washing operation wasperformed, then 10 ml of physiological saline was added to the pellet,and the resulting suspension was used as a live cell suspension. Livecell count was measured by using an L agar plate medium.

Injured cells were prepared by putting 1 ml of the live cell suspensioninto a 1.5-ml microtube, and immersing the microtube in boiling waterfor 50 seconds (injured cell suspension). The injured cells obtained bythis treatment did not form colonies on a standard agar medium.

1-2) EMA Treatment and Light Irradiation Treatment of Bacteria

EMA (Sigma, St. Louis, Mo., USA) was dissolved at 1000 μg/ml usingsterilized water, and subjected to filtration sterilization by using0.20-μm filter (Minisart-plus, Sartorius AG, Gottingen, Germany).

The EMA solution (1000 μg/ml) in a volume of 10 μl was added to the livecell and injured cell suspensions (1 ml) of E. coli DH5α (7.91±0.20log₁₀ cells/ml), and left at 4° C. for 10 minutes under light shielding.

Then, the suspensions were placed at a distance of 20 cm from a visiblelight source (100V PRF 500 W Flood eye, Iwasaki Electric Co., Ltd.,Tokyo, Japan), and irradiated with light for 5 minutes on ice.

Each EMA-treated sample was subjected to refrigerated centrifugation at4° C. and 15,000×G for 10 minutes, the supernatant was removed, and thena similar washing operation was performed with 1 ml of physiologicalsaline.

The live cell suspensions and the injured cell suspensions of theSalmonella enteritidis IID604 (8.47±0.02 log₁₀ cells/ml), Klebsiellaoxytoca JCM1665 (8.38±0.08 log₁₀ cells/ml), and Citrobacter koseriJCM1658 (8.02±0.06 log₁₀ cells/ml) were also subjected to the same EMAtreatment as that used for the E. coli DH5α.

1-3) Extraction of DNA from Coliform Bacteria (IncludingEnterobacteriaceae Bacteria)

The supernatant of each suspension obtained after the EMA treatment wasremoved, then 0.5 ml of 10 mM Tris-HCl (pH 8.0) was added to theprecipitates (cells), 10 μl of a Proteinase K solution (corresponding to1,250 U/ml, Sigma) was added to the mixture, 200 μl of a 10% (w/v) SDSsolution was added to the mixture, and the mixture was incubatedovernight at 50° C.

Then, DNA extraction was performed by the phenol/chloroform extractionand ethanol precipitation method (EP).

Sterilized water in a volume of 150 μl was added to the extracted andpurified DNA, and the concentration was estimated on the basis of theabsorbance at UV 260 nm (OD₂₆₀). Further, purity was estimated on thebasis of OD₂₆₀/OD₂₈₀.

1-4) Real-Time PCR

By using Primer F: forward primer 16S_10F for 16S rRNA gene detection(SEQ ID NO: 1), and Primer R: reverse primer 16S_1500R for 16S rRNA genedetection (SEQ ID NO: 2), a PCR buffer having the composition shownbelow was prepared.

-   a) Ex-Taq (5 U/μl, Takara-Bio): 0.5 μl-   b) 10×Ex-Taq Buffer (Takara-Bio): 5 μl-   c) dNTP mixture (Takara-Bio): 4 μl-   d) Primer F (10 pmol/μl): 4 μl-   e) Primer R (10 pmol/μl): 4 μl-   f) SYBR Green I (2×) (BMA): 10 μl-   g) Sterilized water: 22.5 μl

The template DNA in an amount equivalent to 150 ng was added to 50 μl ofthe aforementioned PCR buffer, and real-time PCR was performed accordingto the following PCR thermal cycle conditions by using a real-time PCRapparatus (iCycler iQ, Bio-Rad, Hercules, Calif.).

-   1) 4° C. for 3 minutes (1 cycle)-   2) 94° C. for 30 seconds (1 cycle)-   3) 94° C. for 20 seconds; 55° C. for 30 seconds; 72° C. for 90    seconds (50 cycles)-   4) 95° C. for 3 minutes (1 cycle)

Then, according to the protocol of the melt analysis of the PCRamplified product (temperature was raised at intervals of 0.1° C. from60° C., each temperature was maintained for 8 seconds, and thisprocedure was repeated 350 times in total up to the final temperature of95° C.), the melting temperature of the PCR amplified product wasmeasured.

1-5) Gel Electrophoresis

2% Agarose gel (2% Seakem GTG agarose, FCM BioProducts, Rockland, Me.)was prepared by using 0.5×TAE.

The PCR amplified product was applied in a volume of 10 μl to theagarose gel, and electrophoresis was performed.

The gel was stained with 1 μg/ml ethidium bromide solution, and observedas a densitograph, and the image thereof was captured and stored byusing AE-6905H Image Saver HR (Atto Co., Japan).

2. Results

Ct values (number of PCR cycles at which the amplification curve exceedsa limit value) obtained by performing real-time PCR are shown in Table4. Further, the results of the electrophoresis are shown in FIG. 3.

The meanings of the symbols used in FIG. 3 are as follows.

-   Klebsiella bacterium: K. oxytoca JCM1665 (8.38±0.08 log₁₀ cells/ml)-   Citrobacter bacterium: C. koseri JCM1658 (8.02±0.06 log₁₀ cells/ml)-   Escherichia bacterium: E. coli DH5α (7.91±0.20 log₁₀ cells/ml)-   Salmonella bacterium: S. enteritidis IIP604 (8.47±0.02 log₁₀    cells/ml)-   EMA+: EMA treatment (10 μg/ml, 10 minutes, 4° C. under light    shielding)+visible light irradiation (5 minutes)-   EMA−: No EMA treatment-   NC: Negative control in which sterilized water was used instead of    the DNA template-   M: 100 bp DNA ladder-   Injured cell: Injured cells prepared by immersing the live cell    suspension in boiling water for 50 seconds.

TABLE 4 Live cell ^(a)) Injured cell ^(b)) Bacrerium log₁₀ cells/ml 0^(c)) 10 ^(d)) 0 ^(e)) 10 Klebsiella oxytoca JCM1665 8.38 ± 0.08 21 ±0.9 22 ± 1.1 22 ± 1.0 n.d. ^(f)) Citrobacter koseri JCM1658 8.02 ± 0.0619 ± 0.7 21 ± 0.8 20 ± 1.1 n.d. Escherichia coli DH5 α 7.91 ± 0.20 24 ±1.2 25 ± 0.8 22 ± 1.4 40 ± 1.4 Salmonella enteritidis IIP604 8.47 ± 0.0221 ± 1.4 21 ± 0.5 20 ± 0.8 34 ± 1.1 The symbols ^(a)) to ^(f)) used inTable 4 indicate the followings. ^(a)) Live cell counts of theKlebsiella bacterium, Citrobacter bacterium, Escherichia bacterium, andSalmonella bacterium numerical values in the columns mean Ct valuesobtained in real-time PCR. ^(b)) Injured cells prepared by immersing thelive cell suspension in boiling water for 50 seconds. ^(c)) No treatmentwith EMA is meant. ^(d)) EMA final concentration of 10 μg/ml is meant.^(e)) Ct value is indicated as mean ± SD (n = 2). ^(f)) n.d. means thatthe PCR amplification reaction did not advance, and the Ct value couldnot be observed.

According to the results shown in Table 4, significant change of the Ctvalue was not provided in the real-time PCR by performing the EMAtreatment for the live cells of E. coli and S. enteritidis. Further, inthe case of the injured cells, the EMA-treated E. coli cells showed ahigher Ct value of about 18, the EMA-treated S. enteritidis cells showeda higher Ct value of about 14, compared with those of the untreatedcells, thus there was observed a tendency that the PCR amplification wassuppressed, but PCR showed a positive reaction (Ct value: 40±1.4 and34±1.1).

As seen from the results of distinction of live cells and injured cellsusing the final PCR amplified products (FIG. 3), the band of theobjective gene was obtained also in the sample of EMA-treated injuredcells for E. coli DH5α and S. enteritidis IID604, and thus distinctionof live cells and injured cells could not sufficiently be confirmed.

On the other hand, as for the Klebsiella bacterium and the Citrobacterbacterium, when the live cells were subjected to the EMA treatment, anyphenomenon concerning significant increase of the Ct value was notobserved, but when the injured cells were subjected to the EMAtreatment, the PCR amplification reaction was completely inhibited, thusthe Ct value could not be measured, and therefore distinction of thelive cells and injured cells was possible.

Example 4

It was examined how much degree the cells of Enterobacter sakazakii werelysed (lysis) in the presence of a pretreatment agent after 50 times ofPCR thermal cycles.

1. Test Methods

Cells of Enterobacter Sakazakii ATCC51329 strain (ES) in an amount of10⁸ cells/ml were suspended in physiological saline or the pretreatmentagent solution mentioned in Table 5 (henceforth also referred to as “DB(direct buffer)”) to prepare suspensions (0.25 mL). Each suspension wasdivided into 25 μl portions, and they were put into 200-μl PCR tubes,respectively, subjected to PCR thermal cycle repetition step using acycle of 95° C. for 15 seconds, 60° C. for 20 seconds, and 72° C. 30seconds (50 times), and combined into one again (total 0.25 ml) as asample for PCR amplification. From the sample of 0.25 ml mentionedabove, a portion of 2.5 μl was collected, and added to 12.25 μl of thepretreatment agent solution mentioned in Table 5 (provided that thevolume of sterilized water was changed to 2.7 μl), and 12.75 μl of thePCR buffer mentioned below was added to the mixture to perform PCR(corresponding to the suspension I mentioned in Table 6). As theprimers, ompA_F: forward primer for ompA gene detection(5′-ggatttaaccgtgaacttttcc-3′, SEQ ID NO: 7), and ompA_R: reverse primerfor ompA gene detection (5′-cgccagcgatgttagaaga-3′, SEQ ID NO: 8) wereused.

Composition of PCR buffer:

-   a) ompA_F (10 pmol/μl): 2 μl-   b) ompA_R (10 pmol/μl): 2 μl-   c) Ex-Taq (5 U/μl, Takara-Bio): 0.25 μl-   (containing 0.5% Tween 20, 0.5% Nonidet P-40, and 50% glycerol)-   d) 10×Ex-Taq Buffer (Takara-Bio): 2.5 μl-   e) dNTP mixture (Takara-Bio): 2 μl-   f) 10×SYBR Green I (BMA): 4 μl

Then, 247.5 μl of the remainder sample for PCR amplification (the samplefor PCR amplification after the 2.5 μl portion was extracted from thevolume of 0.25 ml) was subjected to refrigerated centrifugation(10,000×g, 5 minutes, 4° C.), and 12.25 μl of the pretreatment agentsolution and 12.75 μl of the PCR buffer were added to 2.5 μl of thesupernatant to perform PCR (supernatant I). Then, to the pellet obtainedby the aforementioned centrifugation, 0.25 ml of physiological saline orthe pretreatment agent solution mentioned in Table 5 was added toprepare suspensions, and to 2.5 μl of each suspension, 12.25 μl of thepretreatment agent solution and 12.75 μl of the PCR buffer were added toperform PCR (suspension II). The remainder of the suspension wassubjected to refrigerated centrifugation under the same conditions asmentioned above, and to 2.5 μl of the supernatant, 12.25 μl of thepretreatment agent solution mentioned in Table 5 and 12.75 μl of the PCRbuffer were added to perform PCR (supernatant II). Thereafter, the sameprocedure was repeated until the suspension IV and the supernatant IVwere obtained, and PCR was performed with each of them.

Real-time PCR was performed according to the following PCR thermal cycleconditions by using a real-time PCR apparatus (iCycler iQ, Bio-Rad,Hercules, Calif.).

-   1) 4° C. for 3 minutes (1 cycle)-   2) 95° C. for 15 seconds; 60° C. for 20 seconds; 72° C. for 30    seconds (50 cycles)-   3) 95° C. for 3 minutes (1 cycle)

Then, according to the protocol of the melt analysis of the PCRamplified product (temperature was raised at intervals of 0.1° C. from60° C., each temperature was maintained for 8 seconds, and thisprocedure was repeated 350 times in total up to the final temperature of95° C.), the melting temperature of the PCR amplified product wasmeasured.

TABLE 5 Pretreatment agent Final solution (DB) Amount (μL) concentration500 μg/mL lysozyme 0.5 10 μg/mL 4% Brij 58 6.25   1% 2% BSA 2.5 0.2% 250mM TSC 0.1 1 mM 750 mM MgCl₂ 0.1 3 mM 320 X SYBR Green I 0.1 1.28 XSterilized water 15.45 — Total 25 —2. Results

The results for the Ct values determined by the real-time PCRamplification are shown in Table 6. The indication “Not heated” in thetable means a group for which the heat treatment according to the PCRthermal cycle (repeating 50 times a thermal cycle consisting ofreactions at 95° C., 60° C. and 72° C.) was not performed, and “Heatedwith thermal cycle” means a group for which the heat treatment accordingto the PCR thermal cycle was performed 50 times. The live cell count ofEnterobacter sakazakii in the suspension IV indicated with a) was10^(7.6) cells/ml as determined on the standard agar medium plate, andthe live cell count of Enterobacter sakazakii in the supernatant Iindicated with b) was 10^(5.7) cells/ml as determined by the samemethod.

On the basis of the fundamental characteristic of this test, themeasurement results of live cell counts in the suspension IV andsupernatant I revealed that live cells were also contained at a ratio of1% in the supernatant obtained by refrigerated centrifugation of thesuspension of Enterobacter sakazakii in physiological saline. As for thenon-heated group of E. sakazakii (in physiological saline), when the Ctvalues obtained for the suspension I or II and the supernatant I werecompared, the Ct values for the suspension group were lower by about 5,and it is considered that this is mainly because the live cells weresignificantly collected in the precipitates (pellet), and an extremelysmall amount of them were also collected in the supernatant. That is,such a phenomenon means that the live cells were distributed between theprecipitates and the supernatant at a specific ratio.

The results for the thermal cycle heated group of E. sakazakii (inphysiological saline) means the test results obtained by subjecting thesuspension of the live cells of Enterobacter sakazakii in physiologicalsaline to the PCR thermal cycle 50 times, and then performing PCRamplification reaction with the supernatant and the pellet, and if thebacterial cells were lysed when the PCR thermal cycle was repeated, andthe chromosomal DNA flew mainly out of the cells, it should basicallybecome impossible to measure the Ct value for the suspensions II to IV.However, all of the Ct values were lower than 20, and thus the reactionsof PCR were favorably performed. Thus, it was decided to determine howmuch degree Enterobacter sakazakii cells were lysed after they weresubjected to 50 times of the PCR thermal cycles in physiological salinein the following examples.

TABLE 6 Washing step I II III IV E sakazakii Not Suspension 16.4 ± 0.417.0 ± 0.6 17.0 ± 0.8 16.9 ± 0.5^(a)) (in heated Supernatant 21.5 ±0.0^(b)) 21.2 ± 0.5 23.2 ± 1.0 23.9 ± 1.2 physiological HeatedSuspension 14.3 ± 0.5 16.9 ± 0.4 18.0 ± 1.1 19.0 ± 0.8 saline) withSupernatant 15.2 ± 0.2 19.6 ± 0.6 20.6 ± 0.9 20.8 ± 12 (10⁸ cells/mL)thermal cycle E sakazakii Not Suspension 15.8 ± 1.4 15.4 ± 0.7 15.7 ±1.2 14.3 ± 0.9 (in DB) heated Supernatant 23.9 ± 2.7 27.1 ± 0.8 25.5 ±5.9 28.0 ± 1.4 (10⁸ cells/mL) Heated Suspension 22.4 ± 0.5 22.5 ± 0.422.4 ± 0.4 22.6 ± 0.2 with Supernatant 30.7 ± 0.4 30.9 ± 0.8 30.7 ± 1.030.8 ± 12 thermal cycle

Further, as for the Enterobacter sakazakii cells “not heated” in thepresence of the pretreatment agent, the ratio of the Enterobactersakazakii cells flown into the supernatant by the refrigeratedcentrifugation was suppressed 10 times compared with that obtained withphysiological saline, i.e., the ratio was 0.1 to 0.2%, and the recoveryefficiency as the pellet was markedly improved. If the Enterobactersakazakii cells are lysed only by leaving them in the pretreatment agentsolution, the Ct value significantly increases as the process proceedsfrom the step for the suspension I to the step for the suspension IV,and if they are completely lysed, the chromosomal DNA is recovered inthe supernatant after the refrigerated centrifugation, and thus themeasurement of the Ct value should become impossible. However, theresults shown in Table 6 do not support such estimation.

What should be especially noted for the case where the Enterobactersakazakii cells were subjected to 50 times of PCR thermal cycles in thepretreatment agent solution includes that the Ct value of the suspensionI was smaller than the Ct value of the supernatant I by more than 8,that the Ct value of the suspension II is similarly smaller than the Ctvalue of the supernatant I by more than 8, and that the Ct values of thesuspensions III and IV did not increase in spite of the increase of thenumber of washing operation of the pellet. If the Enterobacter sakazakiicells are completely lysed in the pretreatment agent solution during therepetition of the PCR thermal cycle, and the chromosomal DNA iscompletely flown out into the external solution, the Ct values of thesuspension I and the supernatant I should be substantially the samevalues, and as the number of the washing of the pellet increasesthereafter, the Ct value of the suspension should significantlyincrease, or the measurement thereof should become impossible. However,the results shown in Table 6 do not support such estimation. On thecontrary, the Ct value of the suspension I was smaller than the Ct valueof the supernatant I by more than 8, it is estimated that the ratio ofthe chromosomal DNA flown out into the supernatant was around 0.1 to0.5% at most (as for the origin of Ct value of the supernatant I, anextremely small part of the Enterobacter sakazakii cells of whichspecific gravity became smaller due to the repetition of the thermalcycle may be recovered in the supernatant), and it is estimated that 99%or more of the origin of the Ct value of the suspension I is DNA in thecells of Enterobacter sakazakii. That is, it was suggested that evenafter the Enterobacter sakazakii cells were subjected to the PCR thermalcycle 50 times in the presence of the pretreatment agent, 99% or more ofthem were not lysed.

Further, the Ct values of the suspension I and the supernatant I of the“Not heated” group of E. sakazakii (in DB) shown in Table 6 aresignificantly smaller than those of the “Heated with thermal cycle”group, respectively. Supposing that most of the cells of Enterobactersakazakii were lysed by 50 times of PCR thermal cycles in the presenceof the pretreatment agent, and the chromosomal DNA moved to the solutionside, the Ct values of the suspension I and the supernatant I of the“Heated with thermal cycle” group should be comparable. Further, in thesuspension II, since Enterobacter sakazakii cells were in a state thatthey did not have the chromosomes, and therefore the cells should cometo the state that the Ct value could not be measured. However, theresults shown in Table 6 do not support such estimation. Furthermore, onthe hypothesis that the Ct values of the aforementioned suspension I andthe supernatant I in the “Not heated” group significantly smaller thanthose of the “Heated with thermal cycle” group were provided bydenaturation due to the 50 times of the PCR thermal cycles, becauseproteins such as egg white lysozyme and bovine serum albumin werecontained in the pretreatment agent, the experiment shown in Table 7 wasfurther performed. The specific experimental procedure is shown below.

Ten portions of 25 μl of the pretreatment agent solution having thecomposition shown in Table 5 were prepared, subjected to 50 times of PCRthermal cycles, and combined to prepare 250 μl of the pretreatment agentsolution subjected to the heating with thermal cycle. Then, a pellet oflive cells (washed) of the Enterobacter sakazakii ATCC51329 strainobtained from 50 μl of a culture broth in which the cells wereprecultured overnight was added to physiological saline, a pretreatmentagent solution or a pretreatment agent solution subjected to the heatingwith thermal cycle, at a density of 10⁶ to 10⁹ cells/ml, and suspendedtherein (50 μl of each suspension was prepared). Each suspension in avolume of 2.5 μl was added to 12.25 μl of the pretreatment agentsolution mentioned in Table 5 (provided that the volume of sterilizedwater was changed to 2.7 μl), and 12.75 μl of the PCR buffer was furtheradded to perform PCR (measurement was performed twice). The results areshown in Table 7.

TABLE 7 Pretreatment agent solution (DB) E. sakazakii live PhysiologicalHeated with cell (cells/mL) saline Not heated thermal cycle 10⁹ 16.8 ±0.5 15.8 ± 0.4 16.8 ± 0.8 10⁸ 17.4 ± 0.7 16.2 ± 0.3 15.5 ± 0.7 10⁷ 19.9± 0.2 19.9 ± 0.5 20.6 ± 0.6 10⁶ 22.7 ± 1.1 24.0 ± 0.7 24.0 ± 0.8

It cannot be considered that the Ct value of the live cells ofEnterobacter sakazakii suspended in the pretreatment agent solution andsubjected to the heating with thermal cycle was significantly largerthan the Ct value obtained with physiological saline or the pretreatmentagent solution not subjected to the repetition of the thermal cycle, andthus it was confirmed that it could not be the cause of at least theincrease of both the Ct values of the suspension I and the supernatant Iof the “Heated with thermal cycle” group shown in Table 6. On the basisof these results, it is estimated that the chromosomal DNA in the cellsof Enterobacter sakazakii, which was obtained by subjecting theEnterobacter sakazakii cells to 50 times of the PCR thermal cycles inthe presence of the pretreatment agent, cooling them to 4° C., thenreturning them to room temperature, and subjecting them to refrigeratedcentrifugation so as to recover the chromosomal DNA in the pellet andthe supernatant, was intricately twisted around denatured DNA bindingproteins and denaturation enzymes, and could not function as a templatefor PCR at the beginning. Even if the Ct value of the supernatant Ioriginated in that less than 1% of the Enterobacter sakazakii cells werelysed by 50 times of the PCR thermal cycles in the presence of thepretreatment agent, it is thought that DNA was not completely separatedinto single strands in the PCR thermal cycle treatment at 94° C. at thebeginning of PCR for the aforementioned reason.

Example 5

It was evaluated whether the cells of Enterobacter sakazakii were lysed(lysis) by 50 times of PCR thermal cycles in the presence of thepretreatment agent, by using samples obtained before and after therepetition of the PCR thermal cycles on the basis of fluorescencemicroscopy and stereoscopic microscopy using a nuclear staining agent,and flow cytometry, which enables quantification of cells remainingafter the repetition of the PCR thermal cycles.

A. Fluorescence Microscopy and Stereoscopic Microscopy

1. Experimental Methods

In the same manner as that of the method of Example 4, 10⁹ cells/ml ofthe cells of the Enterobacter Sakazakii ATCC51329 strain (ES) weresuspended in physiological saline or the pretreatment agent solutionmentioned in Table 5 to prepare suspensions (0.25 mL). Each suspensionwas divided into 25 μl portions and put into 200 μl PCR tubes,respectively, and subjected to a PCR thermal cycle repetition step (95°C. for 15 seconds, 60° C. for 20 seconds, and 72° C. 30 seconds, 50times), and then the portions were combined into one again (total 0.25ml). Each suspension was divided into halves, and supernatants werecollected from one of them as it was and the other one subjected torefrigerated centrifugation (3000×g, 10 minutes, 4° C.). To 0.125 ml ofeach of the aforementioned suspensions and supernatants that underwenteach process, SYTO9 was added at a ratio of 1.5 μl/ml, the mixture wasmaintained at 4° C. for 15 minutes under light shielding, and 2.5 μl ofthe mixture was placed on slide glass, covered with cover glass, set ona fluorescence/stereoscopic microscope AxiosKop2 motplus (lens:Plan-NEOFLUAR 100×/1.30 oil ∞/0.17, light source: KublercoDIX ebq 100isolated, software: AxioVision Rel. 4.6.3, filter: FITC and DIC3,exposure time: FITC 347 ms fixed; DIC3 20 ms fixed, LEJ Leistungselektronik Jena GmbH, Germany), and observed to confirm whether thebacterial cells emitted green fluorescence of 530 nm with argon laserlight of 488 nm as an excitation light.

2. Results

Fluorescence microscopy images of the suspensions of Enterobactersakazakii in physiological saline not heated or subjected to 50 times ofPCR thermal cycles, and supernatants obtained by refrigeratedcentrifugation thereof, as well as the suspensions of Enterobactersakazakii in the pretreatment agent solution not heated or subjected to50 times of PCR thermal cycles, and supernatants obtained byrefrigerated centrifugation thereof are shown in FIGS. 4 to 11,respectively. That is, the experiments were performed so that thefluorescence microscopy images should correspond to the suspension I tothe supernatant I of the washing step I mentioned in Table 6. With theseimages, stereoscopic microscopy images, and superposed images of thestereoscopic microscopy images and fluorescence microscopy images arealso shown.

Regardless of use or disuse of the thermal cycle step, bacterial cellsof Enterobacter sakazakii were also found in the centrifugationsupernatant of the physiological saline suspension of Enterobactersakazakii, and it also correlated with the results of PCR shown in Table6. As shown in FIGS. 4 and 6, even if the cells of Enterobactersakazakii were subjected to 50 times of the PCR thermal cycles inphysiological saline, most of them maintained the bacterial cellmorphology (stereoscopic microscopy image and fluorescence microscopyimage), clear SYTO9 staining images were obtained, and therefore it wasconsidered that the cells harbored the chromosomal DNA in the cells.Since cell wall debris were not found in the stereoscopic microscopyimage shown in FIG. 6, a possibility was suggested that the smalldifference of the Ct values of the suspension I and the supernatant I ofthe thermal cycle heated group of the physiological saline suspension ofEnterobacter sakazakii shown in Table 6 was not mainly due to flowingout of DNA into the aqueous phase caused by lysis of the cells ofEnterobacter sakazakii, but it was observed because the specific gravityof the Enterobacter sakazakii cells became small due to 50 times of thePCR thermal cycles, and the ratio of the Enterobacter sakazakii cellsrecovered in the supernatant increased (this was also suggested by theresults of the quantification by flow cytometry shown in FIG. 12explained later).

Further, as shown in FIGS. 8 and 10, lysis of the Enterobacter sakazakiicells themselves was not observed after 50 times of the PCR thermalcycles in the presence of the pretreatment agent, as in the case ofphysiological saline suspension, and it was considered that theEnterobacter sakazakii cells harbored the chromosomal DNA in the cells.However, as a marked difference from the case of using physiologicalsaline, there was observed a phenomenon that the Enterobacter sakazakiicells coagulated after 50 times of the PCR thermal cycles were performedin the presence of the pretreatment agent, but lysis of the bacterialcells was not observed.

B. Flow cytometry

1. Experimental Methods

The experimental methods for flow cytometry are shown below. First, inthe same manner as that of the method of Example 4, 10⁹ cells/ml of thecells of the Enterobacter Sakazakii ATCC51329 strain (ES) were suspendedin physiological saline or the pretreatment agent solution mentioned inTable 5 to prepare suspensions (0.25 mL). Each suspension was divided 25μl portions and put into 200 μl PCR tubes, respectively, and subjectedto a PCR thermal cycle repetition step (95° C. for 15 seconds, 60° C.for 20 seconds, and 72° C. 30 seconds, 50 times), and the portions werecombined into one again (total 0.25 ml). Since suspension andsupernatant thereof of each sample are used for the flow cytometry,three portions in a volume of 0.25 ml each were prepared for eachsample. Specifically, the first portion consisted of the sample per se,the second portion was prepared by subjecting the sample to refrigeratedcentrifugation (3000×g, 10 minutes, 4° C.), then removing thesupernatant, and adding 0.25 ml of physiological saline to theprecipitates to suspend the cells therein, and the third portionconsisted of a supernatant obtained by refrigerated centrifugation ofthe sample similar to the above. To each portion, SYTO9 was added at aconcentration of 1.5 μl/ml, and the mixture was stored at 4° C. for 15minutes under light shielding, and used as a test sample for flowcytometry.

The measurement apparatus was FACS Calibur (BECTON DICKINSON), and anargon laser of 488 nm was used to recognize bacterial cell plots by FSC(forward scattering light measurement) and SSC (side scattering lightmeasurement). If SYTO9 intercalated into the intracellular chromosomalDNA, green fluorescence could be detected with an FL1 filter of whichλmax is 530 nm by excitation with that laser, and therefore FL1 plottingwas also performed. Although any nuclear staining agent based onpropidium iodide (PI) was not especially used, red fluorescence was alsomeasured with an FL3 filter for reference. The details of themeasurement conditions of the flow cytometry are shown in Table 8.

TABLE 8 Parameter Detection Voltage Amp-Gain Mode p1 FSC E02 log p2 SSC427 log p3 FL1 542 log p4 FL2 647 log p5 FL3 633 log p6 FL1-A 1.00 linp7 FL1-W 1.00 lin DDM Param FL12. Results

The experimental results for the physiological saline suspension of theEnterobacter sakazakii cells and the supernatant thereof (not heated ortreated with repetition of PCR thermal cycles) are shown in FIG. 12, andthe experimental results for the bacterium suspended in the pretreatmentagent solution (including re-suspended suspension after washing once)and the supernatant thereof (not heated or treated with repetition ofPCR thermal cycles) are shown in FIG. 13. As for the physiologicalsaline suspension of the Enterobacter sakazakii cells, if most of thecells were lysed by 50 times of the PCR thermal cycles, they weredivided into small portions, and were not included in the bacterium gateregion (the region surrounded by the polygon is a region where bacteriaare plotted) of the FSC-SSC chart, and nothing was plotted in the FL1(FL1-H in the drawing) positive (+) region (right half field withrespect to the X-axis), which means green fluorescence by SYTO9thereafter, or plots therein should markedly decrease. However, theresults for the samples of the physiological saline suspension ofEnterobacter sakazakii cells not heated or subjected to the repetitionof PCR thermal cycles shown in FIG. 12 do not support such estimation.On the contrary, even from the simple comparison of numerical values, itis estimated that 95% of the cells maintained bacterial morphology evenafter the thermal cycles, and also harbored the chromosomal DNA. If theintrinsic standard deviation for the results of two or more times offlow cytometry measurements is taken into consideration, the numericaldifference is highly possibly within the range of measurement error, andit can be considered that substantially 100% of the bacterial cells ofEnterobacter sakazakii maintained the morphology after 50 times of thePCR thermal cycles, and harbored the chromosomal DNA.

Similarly, on the basis of the results for Enterobacter sakazakii cellssuspended in the pretreatment agent solution (not heated or treated withrepetition of PCR thermal cycles) and the suspension obtained by washingand re-suspending the cells (not heated or subjected to PCR thermalcycles) of the Enterobacter sakazakii cells, shown in FIG. 13, it ishard to consider that the bacterial cells of Enterobacter sakazakii werelysed in the presence the pretreatment agent. On the basis of comparisonof the results shown in FIGS. 12 and 13, it is easily estimated that theSYTO9 staining is somewhat inhibited in the presence of the pretreatmentagent. Therefore, it is appropriate that the plots for Enterobactersakazakii suspended in the pretreatment agent solution (not heated orsubjected to thermal cycles, dispersed cells) at an FL1-H intensity(SYTO9) of 10¹ to 10³ shown in FIG. 13 are regarded as the plots derivedfrom the Enterobacter sakazakii cells. On such a premise, it is obviousthat the bacterial cells of Enterobacter sakazakii were not lysed on thebasis of the data for Enterobacter sakazakii cells suspended in thepretreatment agent solution not heated or subjected to PCR thermalcycles, but as for the data for Enterobacter sakazakii cells suspendedin the pretreatment agent solution (obtained by washing andre-suspending the cells) not heated or subjected to PCR thermal cycles,supplementation is needed. In such a case, as a major premise, since theEnterobacter sakazakii SYTO9 staining plots were obtained for the cellsonce washed and re-suspended in physiological saline, they must beevaluated by using the FL1-H (+) region. In this case, although thetotal of the numbers of plots in the FL1-H(+) region for the “dispersedcells” and “coagulated cells” after the PCR thermal cycles apparentlystill smaller than that for the cells not heated, if it is taken intoconsideration that 1 plot of the “coagulated Enterobacter sakazakiicells” is considered to highly possibly consist of at least several toseveral tens of Enterobacter sakazakii bacterial cells as estimated fromthe result shown in FIG. 10, it is inferred that the number isequivalent to or higher than that for the cells not heated, and if thedata obtained without washing are also taken into consideration incombination, it is considered that substantially 100% of the bacterialcells of Enterobacter sakazakii were not lysed.

Example 6

Real-Time PCR Measurement Using Number of Bacterial Cells ofEnterobacter sakazakii and Purified Chromosomes in the Same Amount ofChromosomal DNA Contained in the Cells

1. Experimental Methods

From culture broths of Enterobacter sakazakii ATCC29544 and ATCC51329strains in which the cells were proliferated overnight, purified DNAfree from contamination of RNA was obtained according to the DNAextraction method described in WO2007/094077, absorbance values thereofwere measured at 260 nm and 280 nm (0D₂₆₀ and OD₂₈₀, OD₂₆₀=1.0 for 50μg/ml DNA solution, cell length: 1 cm), the DNA concentration wascalculated from OD₂₆₀, and purity of the purified DNA was estimated onthe basis of OD₂₆₀/OD₂₈₀.

Furthermore, the cells of the aforementioned culture broths in which thecells were proliferated overnight were washed, and then serially dilutedwith sterilized water to prepare live cell suspensions of Enterobactersakazakii at densities of 4×10³ to 4×10⁸ cells/ml. Then, according tothe method of Example 4, 2.5 μl of each suspension was added to 12.25 μlof the pretreatment agent solution mentioned in Table 5 (provided thatthe volume of sterilized water was changed to 2.7 μl), and 12.75 μl ofthe PCR buffer for detection of ompA gene was further added to themixture to perform PCR in the same manner as that of Example 4. Each PCRtube contained 10′ to 10⁶ cells of Enterobacter sakazakii. Since theamount of chromosomal DNA obtained from 1 cell of Enterobacter sakazakiican be regarded 5 fg (5×10⁻¹⁵ g), the amount of chromosomal DNAcontained in each PCR tube was calculated by using that value, and thesame amount of the aforementioned purified DNA (2.5 μl) was put intoeach PCR tube, and the pretreatment agent solution and the PCR bufferwere successively added in the same manner to perform PCR.

2. Results

Purification degrees of DNA are shown in Table 9, and the results ofreal-time PCR are shown in Table 10. As seen from the results shown inTable 9, the values of OD₂₆₀/OD₂₈₀ were around 2.0, and therefore highpurity DNA with little contamination of RNA could be prepared from eachof the two strains of Enterobacter sakazakii, respectively. Further, asseen from the results shown in Table 10, no significant difference wasseen in the Ct values for bacterial cells of Enterobacter sakazakiibacterium and the chromosomal DNA in amounts corresponding to the samenumber of the cells, and thus it was found that if 100% of the purifiedDNA dissolved in the tube functioned as a template of PCR, 100% of thechromosomal DNA of the bacterial cells of Enterobacter sakazakii alsofunctioned as a template for PCR.

As for the case of suspending the cells of Enterobacter sakazakii in asimilar pretreatment agent solution or physiological saline (orsterilized water) and then adding such a PCR buffer as shown in Example4 to the suspension to perform PCR, it is inferred that there may be amisunderstanding according to conventional knowledge that a part of thecells of Enterobacter sakazakii are lysed so that the chromosomal DNAflows out into the external solution, and serves as a template of PCR tocause the reactions of PCR. However, if this hypothesis is applied tothis case, it is necessary that substantially 100% of the Enterobactersakazakii cells should be lysed, but it is obvious that such a 100%lysis phenomenon is denied by the experimental results of Examples 4 and5. That is, as for Example 4, in the comparison of the Ct values of thesuspensions I and II and the supernatant I of the Enterobacter sakazakiicells after 50 times of the PCR thermal cycles in the presence of thepretreatment agent shown in Table 6, if 100% of the cells were lysed,the Ct value of the supernatant I needs to be significantly smaller thanthe Ct value of the suspension II, and the Ct value of this supernatantshould be equivalent to that of the suspension I. However, the resultsshown in Table 6 do not support such estimation. Further, as for Example5, on the basis of the results of the quantification of cell numbers ofEnterobacter sakazakii before and after the repetition of the PCRthermal cycles shown in FIG. 13, which are the results of the flowcytometry measurement, lysis of substantially 100% of the cells isimpossible, and even lysis of 10% of the cells is also improbable. Thatis, the hypothesis that “a part of bacterial cells are lysed, andchromosomal DNA flows out therefrom to the external solution to inducePCR” according to the general scientific knowledge cannot be applied toat least PCR in the presence of the pretreatment agent (50 times)according to the present invention.

TABLE 9 Purified DNA of E. sakazakii after RNAse treatment CD₂₆₀ CD₂₈₀CD₂₆₀/CD₂₈₀ ATCC29544 0.1108 0.0538 2.0595 ATCC51329 0.1082 0.05402.0037

TABLE 10 E sakazakii live cell (cells/PCR tube) 10⁶ 10⁵ 10⁴ 10³ 10² 10¹ATCC29544 DNA 21.2 ± 0.6³⁾ 24.0 ± 0.9 27.1 ± 0.8 31.2 ± 0.9 34.2 ± 1.641.3 ± 5.4 Cell 20.7 ± 0.2 23.9 ± 0.1 27.7 ± 0.1 31.0 ± 0.1 34.3 ± 0.438.3 ± 0.8 ATCC51329 DNA 19.1 ± 0.8 22.1 ± 0.9 25.3 ± 1.0 29.5 ± 1.132.5 ± 1.4 37.3 ± 2.1 Cell 17.2 ± 0.5 20.6 ± 0.4 24.9 ± 0.3 28.6 ± 0.132.1 ± 0.8 37.1 ± 2.0

Example 7

From the results of Examples 4 to 6, it was found that most of theEnterobacter sakazakii cells were not lysed even after the reactions ofPCR (50 times) with the PCR buffer in the presence of the pretreatmentagent, and the cells harbored the chromosomal DNA in the cells. On theother hand, in the TM pattern analysis of the PCR amplified productsafter the real-time PCR (melting temperature measurement), a thermalpeak estimated to be that of the ompA gene product was obtained, and ithad judged to be positive for the real-time PCR. However, to be precise,it was not certain whether the PCR amplified product existed in thebacterial cells, in the reaction solution for PCR, or in the both. Froma commonsense point of view, it is considered that the PCR amplifiedproduct is mainly dissolved in the solution for PCR, but even this isnot definitely clarified, and for the PCR in the presence of thepretreatment agent according to the present invention, it was still lessclarified. In the aforementioned example, it is suggested that the PCRin the presence of the pretreatment agent might be carried out in thebacterial cells, but in the following examples, a possibility that thePCR amplified product remains also in the bacterial cells will bedemonstrated.

1. Experimental Methods

Five portions of 500 μl of culture broth of the Enterobacter sakazakiiATCC51329 strain (9.3×10⁸ cells/ml) in which the cells were proliferatedovernight were prepared, and subjected to refrigerated centrifugation(3000×g, 10 minutes, 4° C.), the supernatant was removed, and then 500μl of a common fixation solution A for bacteria (4% paraformaldehyde),or a fixation solution B (methanol/acetic acid=3/1), fixation solution C(Mildform 10N, 10% formalin, Neutral Buffer Solution Deodorized, WakoPure Chemical Industries, Osaka), or fixation solution D (Mildform 10NM,10% formalin, Neutral Buffer-Methanol Solution Deodorized, Wako PureChemical Industries, Osaka) was added to the pellet, and the mixture wasincubated overnight at 4° C., so that the chromosomal DNA in thebacterial cells and cell wall proteins were crosslinked to fix thechromosomal DNA in the cells beforehand. As a control, a sample in whichthe fixation is not performed was prepared by using 500 μl ofphysiological saline instead of the fixation solution.

Then, the cells were washed three times with 500 μl of physiologicalsaline, and finally suspended in 250 μl of physiological saline. Sincerecovery ratio of bacteria as a pellet after washing operation isgenerally considered 80%, and the centrifugation was performed 4 times,the estimated cell density of Enterobacter sakazakii in the finalpreparation was 7.6×10⁸ cells/ml. Such a suspension in physiologicalsaline in a volume of 250 μl was further diluted 10 times. The estimatedcell density of Enterobacter sakazakii in this dilution was 7.6×10⁷cells/ml. This dilution in a volume of 2.5 μl was used as a sample forPCR amplification, and added to 12.25 μl of the pretreatment agentsolution mentioned in Table 5 (provided that the volume of sterilizedwater was changed to 2.7 μl), and 12.75 μl of the PCR buffer fordetection of gram-negative bacteria mentioned below was further added tothe mixture. Portions in a volume of 27.5 μl were prepared in a numberof 20 for each sample fixed with each fixation solution. As the primers,the forward primer 16S_1234F for 16S rRNA gene detection (SEQ ID NO: 3),and the reverse primer 23S_1703R for 23S rRNA gene detection (SEQ ID NO:4) mentioned in Example 3 were used.

Composition of PCR buffer:

-   a) 16S_1234F (10 pmol/μl): 2 μl-   b) 23S_1703R (10 pmol/μl): 2 μl-   c) Ex-Taq (5 U/μl, Takara-Bio): 0.25 μl    (containing 0.5% Tween 20, 0.5% Nonidet P-40, and 50% glycerol)-   d) 10×Ex-Taq Buffer (Takara-Bio): 2.5 μl-   e) dNTP mixture (Takara-Bio): 2 μl-   f) 10×SYBR Green I (BMA): 4 μl

Real-time PCR was performed according to the following PCR thermal cycleconditions by using a real-time PCR apparatus (iCycler iQ, Bio-Rad,Hercules, Calif.).

-   1) 4° C. for 3 minutes (1 cycle)-   2) 95° C. for 15 seconds; 60° C. for 20 seconds; 72° C. for 3    minutes (30 cycles)-   3) 95° C. for 3 minutes (1 cycle)

Then, according to the protocol of the melt analysis of the PCRamplified product (temperature was raised at intervals of 0.1° C. from60° C., each temperature was maintained for 8 seconds, and thisprocedure was repeated 350 times in total up to the final temperature of95° C.), the melting temperature of the PCR amplified product wasmeasured.

After completion of PCR, the 20 portions of the PCR reaction solutionfor each fixation solution were combined into one, and subjected torefrigerated centrifugation (3000×g, 10 minutes, 4° C.). The supernatantwas collected in a volume of 5 μl and used in 0.8% agarose gelelectrophoresis (SYBR Gold staining, gel staining was performed by SYBRGold staining for all the following cases), and the remainingsupernatant was discarded. To the pellet, 200 μl of physiological salinewas added to suspend the cells. The estimated bacterial cell count ofEnterobacter sakazakii in the suspension was 1.5×10⁷ cells/ml. SYTO9 wasadded to the suspension at a concentration of 1.5 μl/ml, and the mixturewas left at 4° C. for 15 minutes under light shielding, and used toperform flow cytometry under the same conditions as those of Example 5.As a control, the same procedure was performed provided that PCR wasperformed for 0 cycle, not 30 cycle, to obtain a control sample.

Furthermore, for the samples obtained with the fixation solutions A andB, PCR was performed by using the ompA_F primer (SEQ ID NO: 7) and theompA_R primer (SEQ ID NO: 8) described in Example 4 instead of 16S_1234Fand 23S_1703R, the PCR thermal cycle conditions of Example 4, and themethod of Example 7 except for the foregoing conditions, electrophoresiswas performed for 5 μl of the supernatant obtained after PCR, and flowcytometry measurement was performed by using SYTO9 for a physiologicalsaline suspension of the pellet obtained by removing the supernatantafter PCR.

2. Results

The results of the electrophoresis of the reaction supernatants aftereach of the aforementioned fixation solution treatments and PCR (16S-23SrRNA, 2450 bp) are shown in FIG. 14, and the Ct values of thecorresponding reaction solutions determined in the real-time PCR areshown immediately below them. The results of the flow cytometrymeasurement performed by using SYTO9 for suspensions obtained byremoving the supernatants of the samples after PCR, and suspending thepellets in physiological saline are shown in Table 11. Theelectrophoresis images of the supernatants after PCR targeting the ompA(469 bp) gene are similarly shown in FIG. 15. The results of the flowcytometry measurement performed by using SYTO9 for suspensions obtainedby removing the supernatants of the samples after PCR, and suspendingthe pellets in physiological saline are shown in Table 12. As seen fromthe results shown in FIG. 14, the amount of the 16S-23S gene (2450 bp)product in the PCR mixture supernatant obtained with the fixationsolution B was significantly larger than those obtained with the otherfixation solutions, and the band strength was equivalent to thatobtained without fixation (“S” in the drawing). Since, as a standardfixation method of bacteria, use of the fixation solution A is common,and the fixation solutions C and D also have a composition similar tothat of the fixation solution A, it is considered that the chromosomalDNA in the cells of Enterobacter sakazakii and the cell wall proteinswere firmly crosslinked. On the basis of the results shown in FIG. 14,it is appropriate to consider that the fixation solution B may have onlya function equivalent to no fixation (S), but since the fixationsolution B firmly crosslinks mammalian cell chromosomes and cellmembrane proteins, it somewhat exhibits the fixation function also inbacteria.

TABLE 11 FixA 0 cycles Quad Events % Gated % Total FixB 0 cycles QuadEvents % Gated % Total Syto9 UL 0 0.00 0.00 Syto9 UL 0 0.00 0.00 TotalEvents 50000 UR 12 0.15 0.02 Total Events 50000 UR 1 0.03 0.00 GatedEvents 7844 LL 292 3.72 0.58 Gated Events 3761 LL 296 7.87 0.59 LR 754096.12 15.08 LR 3464 92.10 6.93 FixA 30 cycles Quad Events % Gated %Total FixB 30 cycles Quad Events % Gated % Syto9 UL 0 0.00 0.00 Syto9 UL0 0.00 0.00 Total Events 50000 UR 0 0.00 0.00 Total Events 50000 UR 00.00 0.00 Gated Events 7816 LL 1054 13.49 2.11 Gated Events 7905 LL 193024.41 3.86 LR 6762 86.51 13.52 LR 5975 75.59 11.95 FixC 0 cycles QuadEvents % Gated % Total FixD 0 cycles Quad Events % Gated % Total Syto9UL 0 0.00 0.00 Syto9 UL 0 0.00 0.00 Total Events 50000 UR 1 0.01 0.00Total Events 50000 UR 0 0.00 0.00 Gated Events 6873 LL 284 4.13 0.57Gated Events 5785 LL 163 2.82 0.33 LR 6588 95.85 13.18 LR 5622 97.1811.24 FixC 30 cycles Quad Events % Gated % Total FixD 30 cycles QuadEvents % Gated % Total Syto9 UL 0 0.00 0.00 Syto9 UL 0 0.00 0.00 TotalEvents 50000 UR 0 0.00 0.00 Total Events 50000 UR 0 0.00 0.00 GatedEvents 7175 LL 733 10.22 1.47 Gated Events 8726 LL 1845 21.14 3.69 LR6442 89.78 12.88 LR 6881 78.86 13.76 S 0 cycles Quad Events % Gated %Total Syto9 UL 0 0.00 0.00 Total Events 50000 UR 1 0.03 0.00 GatedEvents 3081 LL 1331 43.20 2.66 LR 1749 56.77 3.50 S 30 cycles QuadEvents % Gated % Total Syto9 UL 0 0.00 0.00 Total Events 50000 UR 2 0.040.00 Gated Events 5114 LL 690 13.49 1.38 LR 4422 86.47 8.84

TABLE 12 A 0 cycles Quad Events % Gated % Total B 0 cycles Quad Events %Gated % Total Syto9 UL 0 0.00 0.00 Syto9 UL 0 0.00 0.00 Total Events50000 UR 1 0.01 0.00 Total Events 50000 UR 2 0.01 0.00 Gated Events19549 LL 566 2.90 1.13 Gated Events 16148 LL 5142 31.84 10.28 LR 1898297.10 37.96 LR 11002 68.13 22.00 A 15 cycles Quad Events % Gated % TotalB 15 cycles Quad Events % Gated % Total Syto9 UL 0 0.00 0.00 Syto9 UL 00.00 0.00 Total Events 50000 UR 1 0.01 0.00 Total Events 50000 UR 2 0.010.00 Gated Events 15919 LL 59 0.37 0.12 Gated Events 16803 LL 3730 22.207.46 LR 15859 99.62 31.72 LR 13071 77.79 26.14 A 30 cycles Quad Events %Gated % Total B 30 cycles Quad Events % Gated % Total Syto9 UL 0 0.000.00 Syto9 UL 0 0.00 0.00 Total Events 50000 UR 5 0.03 0.01 Total Events50000 UR 5 0.03 0.01 Gated Events 15707 LL 1563 9.95 3.13 Gated Events14533 LL 1431 9.85 2.86 LR 14139 90.02 28.28 LR 13097 90.12 26.19

The results of the flow cytometry (Quadrant: LR among the 4 quadrants,i.e., comparison of the numbers of bacterial cells of Enterobactersakazakii before and after PCR using the number of SYTO9+ of thebacterium as the index) shown in Table 11 (also Table 12) suggest thateven when the bacterial cells were fixed by using each fixation solutionor without fixation (S) beforehand, namely, the bacterial chromosomalDNA was fixed in the cells, and then PCR thermal cycle was repeated for30 times in the presence of the pretreatment agent, most of thebacterial cells were not lysed, but maintained the morphology, and thechromosomes were retained in the cells. Further, although presence ofthe target gene amplified product also in the PCR reaction mixturesupernatant was suggested by the electrophoresis images (FIG. 14), theCt values determined in the real-time PCR indicated under the gel imagesin FIG. 14 were observed for only the samples obtained with the fixationsolution B and without fixation (S), and Ct value was not observed inreal-time PCR with the other fixation solutions.

It may be considered that this is because, in the cases of the fixationsolutions A, C and D, the fixation degree between the chromosome and thecell wall was high, therefore the thermal denaturation in PCR (95° C.)was not favorably attained, thus the chromosomal DNA could not becomesingle strands, and therefore adhesion of primers to the chromosomebecame poor, but if the results of the similar experiment (30 cycles)targeting the ompA gene (469 bp) shown in FIG. 15 are taken intoconsideration, the Ct values determined in the real-time PCR and bandintensities obtained with the fixation solutions A and B did not showsignificant difference, and therefore the above hypothesis is denied.

That is, it is considered that the small amounts of the target geneamplified products in the PCR reaction mixture supernatants obtainedwith the fixation solution A, C and D shown in FIG. 14 were providedbecause although the PCR reaction itself favorably advanced by using thechromosome harbored by the bacterial cells as a template, the geneproducts were 5 times longer than that of FIG. 15 (2450 bp), and as aresult, flowing out of the gene products amplified in the cells into theexternal solution was suppressed. If this hypothesis is correct, thegene amplified product of 2450 bp should be detected also in thebacterial cells of Enterobacter sakazakii after PCR. This was verifiedin Example 8. As discussed above, it can be concluded that even if atreatment for fixing the bacterial chromosome in the cells to inhibitflowing out of the chromosome into the external solution was performedbeforehand, substantially 100% of the bacterial cells maintained thebacterial morphology, and the chromosome was maintained in the cellseven after repeating a PCR thermal cycle 50 times, so long as it wasperformed in the presence of the pretreatment agent, but in spite ofthat, the reactions of PCR advanced, and the PCR amplified productexisted also in the external solution, and therefore the reactions ofPCR occurred mainly in the bacterial cells. Further, as shown in Example8 mentioned below, a part of the PCR product existed also in the cellsunder the aforementioned conditions.

Example 8

In Examples 4 to 7, it was suggested that the PCR in the presence of thepretreatment agent possibly occurred in the bacterial cells, i.e., insitu PCR possibly occurred. In situ PCR (for example, Gerard J. et al.,American Journal of Pathology, 139: 847-854, 1991) is a technique fordetecting and quantifying a gene such as the HPV gene incorporated intoa chromosomal DNA in human cells, in which before the detection andquantification, human immunocytes are fixed with such a fixationsolution as mentioned in Example 7 to crosslink the chromosomal DNA andhuman cell membrane proteins, and the cells are treated with a proteasefor a short period of time, or cell membranes of the human immunocytesare treated by microwave irradiation.

This technique is a technique of placing a solution for PCR on fixedhuman immunocytes to induce PCR amplification reactions within the humanimmunocytes, with which even a PCR product of about 500 bp does not flowout of the cells. Since the PCR product does not flow out of the cells,if the reactions of PCR are suspended in an early stage of less than 5to 10 cycles, it can be a technique enabling not only detection of agene in cells, but also estimation of the number of incorporated gene.

If PCR in the presence of the pretreatment agent according to thepresent invention is in situ PCR, a part of the amplified product mayremain also in the bacterial cells, and in this example, an experimentwas performed in order to verify it. In Example 7, in particular,although the chromosomal DNA of Enterobacter sakazakii was probablycrosslinked with cell wall proteins with the fixation solution B, therewas observed a tendency that the amplified product of PCR in thepresence to the pretreatment agent more flew out into the externalsolution as compared with the case of using the other fixationsolutions, and a phenomenon similar to that observed with the techniqueof the present invention not using any fixation solution (techniqueusing no fixation (S)).

Therefore, the researches were focused on the fixation solution B and nofixation (S) mentioned in Example 7, and it was examined whether the PCRamplified product would partially remain in the bacterial cells evenwhen a large amount of the PCR amplified product flew out into theexternal solution.

1. Experimental Methods

Two portions of 500 μl of a culture broth of the Enterobacter sakazakiiATCC51329 strain (4.3×10⁸ cells/ml) in which the cells were proliferatedovernight were prepared, and subjected to refrigerated centrifugation(3000×g, 10 minutes, 4° C.), the supernatants were removed, then 500 μlof the fixation solution B (methanol/acetic acid=3/1) was added to eachpellet, the mixture was incubated overnight at 4° C. to crosslink thechromosomal DNA in the bacterial cells and the cell wall proteins, andthereby DNA was fixed in the cells beforehand. As a control, a samplefor which the fixation was not performed was prepared by using 500 μl ofphysiological saline instead of the fixation solution B. The samemethods as those of Example 7 were used thereafter, and the sample wasfinally diluted 10 times to obtain 250 μl of a suspension ofEnterobacter sakazakii in physiological saline. The estimated celldensity of Enterobacter sakazakii in the suspension was about 3.5×10⁷cells/ml. This suspension was used in a volume of 2.5 μl as a sample forPCR amplification, and added to 12.25 μl of the pretreatment agentsolution mentioned in Table 5 (provided that the volume of sterilizedwater was changed to 2.7 μl), and 12.75 μl of the PCR buffer fordetection of gram-negative bacteria mentioned below was added to themixture to perform PCR under the following conditions. At the time ofPCR, portions in a volume of 27.5 μl were prepared in a number of 20 foreach of the sample obtained with the fixation solution and the controlsample. As the primers, the forward primer 16S_1234F for 16S rRNA genedetection (SEQ ID NO: 3), and the reverse primer 23S_1703R for 23S rRNAgene detection (SEQ ID NO: 4) mentioned in Example 3 were used.

After completion of PCR, the 20 portions of the PCR reaction solutionfor each fixation solution were combined into one, and subjected torefrigerated centrifugation (3000×g, 10 minutes, 4° C.). The supernatantwas collected in a volume of 5 μl and used in 0.8% agarose gelelectrophoresis, and the remaining supernatant was discarded. The pelletwas washed twice with 500 μl of physiological saline, and DNA wasextracted by using QuickGene SP kit DNA tissue.

Separately, two 500-μl portions of culture broth in which the cells wereproliferated overnight (4.3×10⁸ cells/ml) were prepared, and subjectedto refrigerated centrifugation (3000×g, 10 minutes, 4° C.), thesupernatants were removed, then 500 μl of the fixation solution B(methanol/acetic acid=3/1) was added to each pellet, and the mixture wasincubated overnight at 4° C. The cells were washed 3 times with 500 μlof physiological saline, and DNA was extracted and purified by usingQuickGene SP kit DNA tissue (Fuji Photo Film Co., Ltd.). As a control, asample in which the fixation was not performed was prepared by using 500μl of physiological saline instead of the fixation solution B. Accordingto the above description, the cell count in the case of applying onlywashing immediately after the fixation and directly extracting DNA fromthe pellet was, since the sample was subjected to centrifugation 4 timesin total, 4.3×10⁸×0.5×0.41=0.9×10⁸ cells, but the bacterial count usedfor PCR was calculated to be about 3.5×10⁷ cells/ml×2.5 μl×20=1.8×10⁶cells in terms of estimated cell density, and if it is taken intoconsideration that the sample was further subjected to centrifugation 3times thereafter, it is estimated to be 0.9×10⁶ cells.

Since the bacterial number used for the DNA extraction step in thesample before PCR was 100 times larger than that in the sample after PCRfor each case, in order to use them in the same cell number, 20 portionseach in a volume of 2.5 μl (=1.8×10⁶ cells) of a physiological salinesuspension of Enterobacter sakazakii (3.5×10⁷ cells/ml) were prepared(fixation solution B and control S), combined into one withoutperforming PCR, then washed 3 times in total by centrifugation, and usedfor the DNA extraction.

Further, even if the long PCR amplified product of about 2450 bp wasconfirmed within the bacterial cells of Enterobacter sakazakii by theaforementioned examination, since the PCR amplified product existed inthe external solution as shown in FIG. 14 or 15 when PCR was performedin the presence of the pretreatment agent, it may be misunderstood thatthe PCR amplified product in the external solution might adsorb on thebacterial cell walls of Enterobacter sakazakii, as if the PCR amplifiedproduct apparently existed in the inside of the cells. Therefore, thefollowing experiment was additionally performed.

Samples were prepared in a number of 20 by adding 2.5 μl of a DNAaqueous solution containing 0.44 ng of DNA (440 pg, the amount ofchromosomal DNA contained in 8.8×10⁴ cells) purified from theEnterobacter sakazakii ATCC51329 strain to each PCR tube, adding apretreatment agent solution as in the aforementioned case, and thenadding a PCR buffer to a total volume of 27.5 μl, and PCR was performedwith each of them under the conditions shown below. Then, the contentsof the 20 PCR tubes were combined into one, 50 μl of a 3.5×10⁷ cells/mlphysiological saline suspension of Enterobacter sakazakii (treated withthe fixation solution B or with no fixation, and washed 3 timesaccording to the method of Example 7) was added to the combined reactionsolution, the mixture was sufficiently mixed, and subjected torefrigerated centrifugation (3000×g, 10 minutes, 4° C.) to remove thesupernatant, and the pellet was washed twice and used for DNAextraction.

Composition of PCR buffer:

-   a) 16S_1234F (10 pmol/μl): 2 μl-   b) 23S_1703R (10 pmol/μl): 2 μl-   c) Ex-Taq (5 U/μl, Takara-Bio): 0.25 μl    (containing 0.5% Tween 20, 0.5% Nonidet P-40, and 50% glycerol)-   d) 10×Ex-Taq Buffer (Takara-Bio): 2.5 μl-   e) dNTP mixture (Takara-Bio): 2 μl-   f) 10×SYBR Green I (BMA): 4 μl

Real-time PCR was performed according to the following PCR thermal cycleconditions by using a real-time PCR apparatus (iCycler iQ, Bio-Rad,Hercules, Calif.).

-   1) 4° C. for 3 minutes (1 cycle)-   2) 95° C. for 15 seconds; 60° C. for 20 seconds; 72° C. for 3    minutes (30 cycles)-   3) 95° C. for 3 minutes (1 cycle)

Then, according to the protocol of the melt analysis of the PCRamplified product (temperature was raised at intervals of 0.1° C. from60° C., each temperature was maintained for 8 seconds, and thisprocedure was repeated 350 times in total up to the final temperature of95° C.), the melting temperature of the PCR amplified product wasmeasured.

2. Results

The results are shown in FIG. 16. The results of electrophoresis ofsupernatants of reaction mixtures obtained in PCR performed in thepresence of the pretreatment agent (16S-23S: 2450 bp) with cells ofEnterobacter sakazakii fixed with the fixation solution B or not fixedare shown in the lanes 2 and 3, the results of electrophoresis of DNAobtained by DNA extraction from the pellet obtained after the PCRmentioned above and washed twice are shown in the lanes 5 and 6, theresults of electrophoresis of DNA directly extracted from the fixed ornot fixed Enterobacter sakazakii cells, which were the test materials ofthis experiment, are shown in the lanes 7 and 8, the results ofelectrophoresis of DNA extracted from the fixed or not fixedEnterobacter sakazakii cells before they were used for the reactions ofPCR are shown in the lanes 9 and 10, and the results of electrophoresisof DNA extracted from the Enterobacter sakazakii cells washed twiceafter addition of the PCR amplified product prepared beforehand areshown in the lanes 13 and 14, respectively.

As shown by the results of the lanes 13 and 14, it was demonstrated thateven if the PCR gene product (2450 bp) adsorbed to Enterobactersakazakii bacterial cell wall outer membranes etc. from the externalsolution side, the PCR gene product could be removed from the bacteriumby washing twice for both the fixed and non-fixed cells. Therefore, thepossibility of incorrect interpretation that the PCR gene product thatmight adsorb on the cell wall outer surfaces was regarded as the PCRgene product remained in the cells was eliminated. Further, by takinginto consideration that a fragment estimated to be the PCR reactionproduct was detected in the lanes 5 and 6 indicating the results for DNAextracted from the cells of which pellet was washed twice after PCR, andthat the fragment was not the PCR gene product that might adsorb on theoutside of the cell wall as seen from the results of the lanes 13 and14, the PCR gene product of the lanes 5 and 6 is highly possibly the PCRgene product remained in the cells and then extracted. Further, even ifthe concentrations of the PCR gene product in the external solution andin the cells became the same, because the cells were bacterial cells ofwhich cell walls were injured, and thus the PCR gene product was in astate that it could freely pass through the cell walls, the volume ofthe external solution was about 10¹⁰ times larger than the volume of thecells, and the amount of the PCR gene product in the external solutionshould also be about 10¹⁰ times larger than that in the cells, that is,1/10¹⁰ of the amount in the external solution should be distributed inthe cells. However, on the basis of comparison of the band intensitiesof the lanes 2, 3, 5 and 6, it cannot be considered that the amount ofthe PCR product remained in the cells was the 1/10¹⁰ amount. That is, itwas suggested that the PCR occurred in the method of the presentinvention might possibly be in situ PCR. As seen from the results of thelanes 7 and 8, the chromosomal DNA was detected from both the fixed andnon-fixed Enterobacter sakazakii bacterial cells used as the testmaterials, but any band of the chromosomal DNA was not obtained in thelanes 5, 6, 9 and 10. This difference was probably provided by thedifference of the cell number of the Enterobacter sakazakii bacterialcells used for the DNA extraction, specifically, the cell number of0.9×10⁶ cells was highly possibly insufficient for the DNA extraction,whereas in the lanes 7 and 8 for the test materials, the cell number wasin fact as high as 0.9×10⁸ cells.

Example 9

The cells of Enterobacter sakazakii were subjected to a boilingtreatment in physiological saline or in the presence of the pretreatmentagent, and it was investigated how much the Enterobacter sakazakiichromosomal DNA was flown out into each supernatant depending on thetreatment time.

1. Experimental Methods

Cells in a culture broth of the Enterobacter sakazakii ATCC51329 strain(1.1×10⁹ cells/ml) in which the cells were proliferated overnight werewashed, and diluted 10 times with physiological saline, and thesuspension was subjected to refrigerated centrifugation (3000×g, 10minutes, 4° C.) to collect a pellet once, an equal volume (500 μl) ofphysiological saline or a pretreatment agent solution having thecomposition shown in Table 5 was added to the pellet, and the cells weresufficiently suspended. Then, the suspension was heated with boilingwater for 0 to 5 minutes, and after the heating, immediately cooled.Each suspension immediately after the heating in a volume of 5 μl and asupernatant obtained by refrigerated centrifugation of each suspensionin a volume of 5 μl were subjected to electrophoresis on 0.8% agarosegel, respectively.

2. Results

In FIG. 17, there are shown degrees of flowing out into the supernatantof the chromosomes of Enterobacter sakazakii subjected to the heattreatment in physiological saline or in the presence of the pretreatmentagent using boiling water. First, as for the results of the lanes 2 and9, although the cells were not heated, the presence of a trace amount ofthe chromosomal DNA was already suggested, but it is considered thatthis was because the cells in the culture broth in which the cells wereproliferated overnight reached the resting stage, therefore a part ofdead cells were lysed, and the chromosomal DNA thereof flew out into theexternal solution. Therefore, such a trace amount of the chromosomal DNAwas ignored in the evaluation. Although it was estimated that DNA flewout in physiological saline from the bacterial cells of Enterobactersakazakii due to the heating, any band of the chromosomal DNA was notdetected for the suspension and the supernatant in the presence of thepretreatment agent even after boiling for 5 minutes. However, whenevaluation was made on the basis of observation of the wells of thelanes 10 and 12, a band was observed in the well in the case of thesuspension, but any band was not observed for the supernatant, and itwas revealed that the chromosomal DNA did not flow out of the bacterialcells in the presence of the pretreatment agent, but remained in thecells. On the other hand, bands were also observed in the wells for thesupernatants of physiological saline (lanes 5, 6 and 7), and it isconsidered that they were derived from a part of dead cells ofEnterobacter sakazakii collected in the supernatant aftercentrifugation, of which specific gravity decreased due to the boiling.The results shown in Table 6 or FIG. 12 also support this estimation.From the above results, it was suggested that, although the conditionsdiffered from those of the repetition of the PCR thermal cycle to beprecise, it is more difficult for DNA to flow out of the bacterial cellsin the presence of the pretreatment agent even when the cells aresubjected to a heat treatment.

INDUSTRIAL APPLICABILITY

According to the method of the present invention, live cells of amicroorganism can be detected with high sensitivity by distinguishingthem from dead cells or injured cells. The present invention enablessimple and quick distinction of live cells, injured cells and dead cellsof a microorganism in foodstuffs, biological samples, swab samples andenvironments such as industrial water, environmental water andwastewater, based on the nucleic acid amplification method. The methodand kit of the present invention can be used for voluntaryinvestigation, and are advantageous also in an economical point of view.

According to a preferred embodiment of the present invention, it canalso be applied to sanitation inspection of various foodstuffscontaining 5 log₁₀ cells/ml or more of injured cells or dead cells ofEscherichia coli, or diagnosis of child bacteriemia in which Escherichiacoli circulates in blood.

Further, according to a preferred embodiment of the present invention,only live cells of coliform bacteria including Enterobacteriaceaebacteria can be detected from foodstuffs with high sensitivity (1CFU/2.22 ml of milk), and more quickly (7 hours and 30 minutes) comparedwith the regulated method (Food Sanitation Law/Ministerial Ordinanceconcerning the Ingredient Standards for Milk and Dairy Products),therefore use thereof is expected for determination before factoryshipments after manufacture in various food factories represented bymilk manufacturing factories, and it is assumed to be highlyindustrially useful.

Furthermore, the present invention enables quick detection andquantification of only live microorganisms at a low concentration, formicroorganisms including not only coliform bacteria andEnterobacteriaceae bacteria, but also various bacteria includingpathogenic bacteria, viruses, and so forth, and therefore it can beapplied to various sanitation inspections, clinical tests, processcontrol, and so forth.

What is claimed is:
 1. A method for detecting live cells of amicroorganism in a test sample by distinguishing the live cells fromdead cells or injured cells, which comprises the steps of: a) adding anagent capable of covalently binding to DNA or RNA by irradiation withlight having a wavelength of 350 nm to 700 nm to the test sample; b)irradiating the test sample to which the agent is added with lighthaving a wavelength of 350 nm to 700 nm; c) amplifying a target regionof DNA or RNA of the microorganism contained in the test sample by anucleic acid amplification method in the presence of (i) lysozyme and/ora nonionic surfactant, (ii) a magnesium salt, (iii) either one of anorganic acid salt and a phosphoric acid salt, and (iv) one or moreselected from the group consisting of albumin, dextran, T4 gene 32protein, acetamide, betaine, dimethyl sulfoxide, formamide, glycerol,polyethylene glycol soybean trypsin inhibitor, α2-macroglobulin,tetramethylammonium chloride, lysozyme, phosphorylase and lactatedehydrogenase without extracting nucleic acids from the cells, and d)analyzing the amplified product, thereby detecting the live cells. 2.The method according to claim 1, wherein, in step c), the amplificationof the target region is performed in the presence of an anionicsurfactant and/or a cationic surfactant.
 3. The method according toclaim 1, wherein, before step c), the steps a) and b) are repeatedlyperformed.
 4. The method according to claim 1, wherein, before step a),the following step a′) is performed: a′) treating the test sample withan enzyme having an activity of decomposing cells other than that of themicroorganism, a colloidal particle of a protein, a lipid or asaccharide in the test sample.
 5. The method according to claim 4,wherein the enzyme is selected from the group consisting of aprotein-degrading enzyme, a lipid-degrading enzyme and asaccharide-degrading enzyme.
 6. The method according to claim 1, whereinthe test sample is selected from the group consisting of a foodstuff, abiological sample, drinking water, industrial water, environmentalwater, wastewater, soil and a swab sample.
 7. The method according toclaim 1, wherein the microorganism is a bacterium or a virus.
 8. Themethod according to claim 7, wherein the bacterium is a gram-negativebacterium.
 9. The method according to claim 1, wherein the agent capableof covalently binding to DNA or RNA by irradiation with light having awavelength of 350 nm to 700 nm is selected from the group consisting ofethidium monoazide, ethidium diazide, propidium monoazide, psoralen,4,5′,8-trimethylpsoralen and 8-methoxypsoralen.
 10. The method accordingto claim 2, wherein the organic acid salt is selected from the groupconsisting of an acetic acid salt, a propionic acid salt and a citricacid salt.
 11. The method according to claim 2, wherein the phosphoricacid salt is a pyrophosphoric acid salt.
 12. The method according toclaim 1, wherein the target region is a target region of 50 to 5,000nucleotides.
 13. The method according to claim 12, wherein the targetregion is a target region corresponding to a gene selected from thegroup consisting of 5S rRNA gene, 16S rRNA gene, 23S rRNA gene and tRNAgene of DNA of the test sample.
 14. The method according to claim 1,wherein the nucleic acid amplification method is PCR, RT-PCR, LAMP, SDA,LCR, TMA, TRC, HC or microarray method.
 15. The method according toclaim 14, wherein PCR is performed as real-time PCR to simultaneouslyconduct PCR and analysis of the amplified product.
 16. The methodaccording to claim 1, wherein the analysis of the amplified product isperformed by using a standard curve representing relationship betweenamount of the microorganism and the amplified product, which is createdby using standard samples of the microorganism.
 17. The method accordingto claim 1, wherein the nonionic surfactant is one or more selected fromthe group consisting of Triton, Nonidet, Tween and Brij.
 18. The methodaccording to claim 1, wherein the amplification of the target region isperformed in microbial cells.